EUROCONFERENCES

The discovery that the biological actions of endothelium-derived relaxing factor (1) are due to the endogenous release of nitric oxide (NO) (2-4) revealed the existence of a ubiquitous biochemical pathway (5). Nitric oxide is formed from the amino acid L-arginine by a family of enzymes, the NO synthases (NOS) which are haem-containing enzymes with a sequence similarity to cytochrome P-450 reductase. Several isoforms of NOS are now known to exist, two of which are constitutive and one of which is inducible by immunological stimuli. The constitutive NOS that was first discovered in the vascular endothelium has been designated as eNOS, whereas that present in the brain, spinal cord and peripheral nervous system is termed nNOS. The form of NOS induced by immunological or inflammatory stimuli is known as iNOS (for reviews see 6-10).

Nitric oxide plays a role in many physiological functions. Its formation in vascular endothelial cells, in response to chemical stimuli and to physical stimuli such as shear stress, maintains a vasodilator tone that is essential for the regulation of blood flow and pressure (see 5). Indeed, mice in which the eNOS gene has been disrupted have an elevated blood pressure compared to wild type animals (11). Nitric oxide produced by the endothelium and/or platelets also inhibits platelet aggregation and adhesion, inhibit leukocyte adhesion and modulates smooth muscle cell proliferation (12). Nitric oxide is synthesized in neurones of the central nervous system, where it acts as a neuromediator with many physiological functions, including the formation of memory, coordination between neuronal activity and blood flow, and modulation of pain (13,14). In the peripheral nervous system, NO is now known to be the mediator released by a widespread network of nerves, previously recognized as nonadrenergic and noncholinergic. These nerves mediate some forms of neurogenic vasodilatation and regulate certain gastrointestinal, respiratory and genitourinary functions (15-17). These physiological actions of NO are mediated by activation of the soluble guanylate cyclase and consequent increase in the concentration of cyclic guanosine monophosphate in target cells (18,19).

In addition, NO is generated in large quantities during host defence and immunological reactions (20,21). Such generation of NO was first observed in activated macrophages (22-24) where it contributes to their cytotoxicity against tumour cells, bacteria, viruses and other invading microorganisms. The cytostatic/cytotoxic actions of NO result from its inhibitor actions on key enzymes in the respiratory chain and in the synthesis of deoxyribonucleic acid in the target cells (25,26). Nitric oxide may also interact with oxygen-derived radicals to produce other toxic substances such as peroxynitrite (27). Thus NO acts both as a physiological mediator and a pathophysiological entity. Recent experiments have shown that NO is a physiological regulator of cytochrome c oxidase and that, in addition, it can irreversibly inhibit other parts of the respiratory cycle, either directly or through the formation of peroxynitrite, leading to cytostasis and cytotoxicity (28,29).

1. FURCHGOTT, R.F. AND ZAWADZKI, J.V. : The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373-376, 1980.

2. PALMER, R.M.J., FERRIGE, A.G. AND MONCADA, S.: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524-526, 1987.

3. IGNARRO, L.J., BUGA, G.M., WOOD, K.S., BYRNS, R.E. AND CHAUDHURI, G.: Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. USA 84:9265-9269, 1987.

4. KHAN, M.T. AND FURCHGOTT, R.F.: Additional evidence that endothelium-derived relaxing factor is nitric oxide. In Pharmacology, edited by M.J. Rand and C. Raper, Elsevier, Amsterdam, pp. 341-344, 1987.

5. MONCADA, S., PALMER, R.M.J. AND HIGGS, E.A.: Biosynthesis of nitric oxide from L-arginine: a pathway for the regulation of cell function and communication. Biochem. Pharmacol. 38:1709-1715, 1989.

6. KNOWLES, R.G. AND MONCADA, S. : Nitric oxide synthases in mammals. Biochem. J. 298:249-258, 1994.

7. MORRIS, S.M. Jr. AND BILLIAR, T.R.: New insights into the regulation of inducible nitric oxide synthesis. Am. J. Physiol. 266: E829-E839, 1994.

8. NATHAN, C. AND XIE, Q.-W.: Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269: 13725-13728, 1994.

9. SESSA, W.C.: The nitric oxide synthase family of proteins. J. Vasc. Res. 31: 131-143, 1994.

10. STUEHR, D.J. AND GRIFFITH, O.W.: Mammalian nitric oxide synthases. Adv. Enzymol. Related Areas. Mol. Biol. 65:287-346, 1992.

11. HUANG, P.L., HUANG, Z., MASHIMO, H., BLOCK, K.D., MOSKOWITZ, M.A., BEVAN, J.A. AND FISHMAN, M.C.; Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature, 377: 239-242, 1995.

12. MONCADA, S. AND HIGGS, E.A.: The L-arginine-nitric oxide pathway. N. Engl. J. Med., 329:2002-2012, 1993.

13. GARTHWAITE, J.: Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci. 14:60-67, 1991.

14. SNYDER, S.H. AND BREDT, D.S.: Biological roles of nitric oxide. Sci. Am. 266: 68-71, 1992.

15. GILLESPIE, J.S., LIU, X. AND MARTIN, W.: The neurotransmitter of the non-adrenergic non-cholinergic inhibitory nerves to smooth muscle of the genital system. In Nitric oxide from L-arginine: a bioregulatory system, edited by S. Moncada and E.A. Higgs, Elsevier Science Publishers B.V., Amsterdam, pp. 147-164, 1990.

16. RAND, M.J.: Nitrergic transmission: nitric oxide as a mediator of non-adrenergic, non-cholinergic neuro-effector transmission. Clin. Exp. Pharmacol. Physiol. 19: 147-169, 1992.

17. TODA, N.: Nitric oxide and the regulation of cerebral arterial tone. In Nitric oxide in the nervous system, edited by S. Vincent, Academic Press Ltd., pp. 207-225, 1995.

18. MURAD, F., ISHII, K., FÖRSTERMANN, U., GORSKY, L., KERWIN, J.F. JR., POLLOCK, J. AND HELLER, M.: EDRF is an intracellular second messenger and autacoid to regulate cyclic GMP synthesis in many cells. Adv. Second Messenger Phosphoprotein Res. 24: 441-448, 1990.

19. IGNARRO, L.J.: Heme-dependent activation of guanylate cyclase by nitric oxide: a novel signal transduction mechanism. Blood Vessels, 28:67-73, 1991.

20. NATHAN, C.F. AND HIBBS, J.B. Jr.: Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr. Opin. Immunol. 3: 65-70, 1991.

21. NUSSLER, A.K. AND BILLIAR, T.R.: Inflammation, immunoregulation and inducible nitric oxide synthase. J. Leukoc. Biol. 54: 171-178, 1993.

22. HIBBS, J.B. JR, TAINTOR, R.R., VAVRIN, Z. AND RACHLIN, E.M.: Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157: 87-94, 1988.

23. MARLETTA, M.A., YOON, P.S., IYENGAR, R., LEAF, C.D. AND WISHNOK, J.S.: Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 27: 8706-8711, 1988.

24. STUEHR, D., GROSS, S., SAKUMA, I., LEVI, R. AND NATHAN, C.; Activated murine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide. J. Exp. Med. 169: 1011-1020, 1989.

25. HIBBS, J.B. JR., TAINTOR, R.R., VAVRIN, Z., GRANGER, D.L., DRAPIER, J.-C., AMBER, I.J. AND LANCASTER, J.R. Jr.: Synthesis of nitric oxide from a terminal guanidino nitrogen atom of L-arginine: a molecular mechanism regulating cellular proliferation that targets intracellular iron. In Nitric oxide from L-arginine: A bioregulatory system, edited by S. Moncada and E.A. Higgs, Elsevier, Amsterdam, pp. 189-223, 1990.

26. NGUYEN, T., BRUNSON, D., CRESPI, C.L., P ENMAN, B.W., WISHNOK, J.S. AND TANNENBAUM, S.R.: DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proc. Natl. Acad. Sci. USA 89:3030-3034, 1992.

27. BECKMAN, J.S., BECKMAN, T.W., CHEN, J., MARSHALL, P.A. AND FREEMAN, B.A.: Apparent hydroxyl radical production by peroxynitrite:implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87:1620-1624, 1990.

*-28. LIZASOAIN, I., MORO, M.A., KNOWLES, R.G., DARLEY-USMAR, V. AND MONCADA, S.; Nitric oxide and peroxynitrite exert distinct effects on mitochondrial respiration which are differentially blocked by glutathione or glucose. Biochem. J. 314, 877-880, 1996.

29. CLEMENTI, E., BROWN, G.C., FEELISCH, M. AND MONCADA, S.; Persistent inhibition of cell respiration by nitric oxide: Crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc. Natl. Acad. Sci. USA, 95, 7631-7636, 1998.

The cytokine-inducible NO synthase (iNOS) participates in host defense by generating NO in response to microbial pathogens, but may also contribute to tissue destruction associated with autoimmune disorders. We believe that understanding iNOS structure could lead

to selective control of its NO synthesis. Moreover, it should helpexplain how enzyme structure relates to cofactor binding andfunction, identify potential paths of electron transfer within theenzyme, and possibly reveal how the enzyme is able to catalyze twodistinct, sequential monooxygenase reactions to convert L-arginine toNO.

All NOS are dimeric, bi-domain polypeptides containing a N-terminaloxygenase domain and a C-terminal reductase domain, with a calmodulinbinding consensus sequence located between the two domains. The mouseiNOS oxygenase domain (amino acids 1-498) contains the dimer interface, the binding sites for L-arginine, heme,tetrahydrobiopterin (BH4), and is the site where NO synthesis occurs.In collaboration with Drs. Tainer and Getzoff at Scripps Research Institute, we have recently published crystal structures of a mouse iNOS oxygenase domain monomer containing a N-terminal deletion (amino acids 1-114) and structures of an iNOS oxygenase dimer containing a smaller N-terminal deletion (amino acids 1-65) (1, 2). The monomer construct contains bound heme but is incapable of binding BH4 and L-arginine or catalyzing NO synthesis, while the dimer construct contains heme, is active, and binds BH4 and substrate with normal affinity (3).

The structures provide many interesting new concepts. For example, the iNOS oxygenase domain is formed by a single continuous fold dominated by a series of overlapping beta sheets which form the center of a flattened molecule. This core structure is surrounded by helices which in some cases display high mobility in the monomer crystal. The high degree of sequence conservation among the NOS oxygenase domains predict that they have this same general structure. The reliance on beta sheet structure clearly differentiates the NOS from the cytochrome P450's, which carry out similar chemistry but are primarily helical. The NOS core beta sheet structure is not changed in the dimer. However, in the dimer movement occurs among discreet helical and loop structures such that they are recruited into the dimer interface to form the BH4 binding sites and active site channel.

Regarding the heme environment, in the NOS monomer the heme is exposed to solvent and is bound to a cysteine thiolate, as predicted by earlier spectroscopic studies. The heme sits on a rim located on the concave side of the monomer with its distal side pointing in toward an extensive hydrophobic pocket. The proximal side of the NOS heme contains aromatic residues that stack with the heme porphyrin ring and in one case hydrogen bond with the cysteine thiolate that coordinates the heme iron. Because similar H-bonding is not observed in P450's, it is suspected to modify NOS heme iron reactivity in ways that favor NO synthesis. In the BH4- and L-arginine-containing dimer, the heme is protected from solvent by L-arginine and by a funnel shaped active site channel that is formed by elements from both oxygenase partners. L-arginine stays positioned above the heme through H-bonding interactions with the carboxylate of E371, consistent with earlier mutagenesis studies that indicated E371 was essential for L-arginine binding to iNOS (4). BH4 is positioned next to the heme with its ring perpendicular to the heme plane. Ring heteroatoms of BH4 form a hydrogen bond network with a heme propionic acid carboxylate and L-arginine. Aromatic residues from both oxygenase subunits engage in stacking interactions with the BH4 ring.

The lack of amino acid proton donors in the distal heme pockets of the dimer suggest that the proton required for oxygen activation in the first step of NO synthesis may be provided by L-arginine itself. A reliance on proton donation from substrate during oxygen activation may help explain how the enzyme can carry out the mechanistically distinct second step involving oxidation of N-hydroxy-L-arginine. Amino acids 65 through approximately 118 form a hairpin loop structure that extends out from its parent subunit in the dimer to interact with the neighboring oxygenase domain. This interaction explains why the hairpin loop structure is essential for dimerization

(3). The two BH4 molecules are positioned near the dimer interface, and structural elements that participate in BH4 binding also make important intersubunit contacts, underscoring an integration between BH4 binding and dimerization in iNOS. One disulfide bond is present in the iNOS dimer and involves the C109 residues of either subunit.

The C109 cysteine is completely conserved among the NOSs and in eNOS may be involved in binding Zn2+ to the enzyme. The C109A mutant of iNOS cannot form the intersubunit disulfide link. It requires a higher BH4 concentration to dimerize or support NO synthesis, but when saturated with BH4 appears to have normal actvity. The entire iNOS dimer interface is extensive and involves many contacts between the two oxygenase components.

There is a conserved electropositive patch on the surface of each oxygenase domain opposite the active site channel, and this patch surrounds a gap where a heme vinyl group comes close to the surface. We propose this to be the site where the reductase domain interacts with the oxygenase and transfers electrons to the heme (1, 2). A comparison of oxygenase monomer and dimer structures suggests that during the process of dimerization a helical element shifts to expose the putative reductase interaction site. This implies that dimerization may cause essential changes in protein structure that allow for electron transfer.

Our related biochemical studies have investigated the reductase-oxygenase interaction and electron transfer pathway. The experiments involve construction of iNOS heterodimers that contain one full-length subunit and one oxygenase domain subunit. Thus, an iNOS heterodimer has two hemes but only one reductase domain and therefore only one path to transfer NADPH-derived electrons to the heme(s). An iNOS heterodimer composed of wild type full-length and oxygenase components was shown to be active for NO synthesis even though only one of its two hemes received electrons from the single reductase domain (5). This suggested that a single reductase domain transfers electrons to only one of the two hemes in the dimer, and that the NOS active sites can function independently of each other. To determine which heme received electrons from the single reductase domain in the heterodimer described above, we constructed a complimentary pair of heterodimers that contained a specific L-arginine binding mutation (E371A) in either the full-length or oxygenase domain subunit, and characterized the proteins regarding L-arginine binding, heme reduction, and NO synthesis (6). The E371A mutation completely prevented L-arginine binding to one oxygenase domain in each heterodimer, but did not affect the L-arginine affinity of its partner subunit, and did not prevent heme iron reduction by the single reductase domain in any case. The mutation prevented NO synthesis when its was present in the oxygenase domain located trans to the single reductase domain, but had no effect when located in the same subunit as the reductase domain. These results imply that the flavin-to-heme electron transfer must proceed exclusively in trans in the heterodimer, rather than within the same subunit. We propose a domain swapping interaction takes place upon dimerization of two full-length iNOS subunits which brings reductase and oxygenase domains from adjacent subunits into contact. This enables intersubunit electron transfer between flavins and heme to take place, resulting in NO synthesis. A reductase-oxygenase domain swapping interaction as we envision for iNOS may have arisen gradually during evolution of the enzyme structure and helps explain why dimerization is essential to activate NO synthesis.

1. Crane, B. R., A. S. Arvai, R. Gachhui, C. Wu, D. K. Ghosh, E. D. Getzoff, D. J. Stuehr, & J. A. Tainer. (1997) The structure of NO synthase oxygenase domain and inhibitor complexes. Science, 278,425-430.

2. Crane, B. R., A. S. Arvai, D. K. Ghosh, C. Wu, E. D. Getzoff, D. J. Stuehr, & J. A. Tainer. (1998) Structure of NO synthase oxygenase dimer with pterin and substrate. Science, 279, 2121-2126.

3. Ghosh, D. K., C. Wu, E. Pitters, M. Moloney, E. R. Werner, B. Mayer, & D. J. Stuehr. (1997) Characterization of the inducible NO synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction. Biochemistry, 36, 10609-10619.

4. Gachhui, R., D. K. Ghosh, C. Wu, J. Parkinson, B. R. Crane, and D. J. Stuehr. (1997) Mutagenesis of acidic residues in the oxygenase domain of inducible NO synthase identifies a glutamate involved in arginine binding. Biochemistry 36, 5097-5103.

5. Siddhanta, U., C. Wu, H. M. Abu-Soud, D. K. Ghosh, & D. J. Stuehr. (1996) Heme iron reduction and catalysis by a NO synthase heterodimer containing one reductase and two oxygenase domains. J. Biol. Chem., 271, 7309-7312.

6. Siddhanta, U., A. Presta, B. Fan, D. Wolan, D. L. Rousseau, & D. J. Stuehr. (1998) Domain swapping in the inducible NO synthase: Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer. J. Biol. Chem. 273, 18950-18958.

Endothelial nitric oxide synthase (eNOS) is a peripheral membrane protein that converts L-arginine to nitric oxide (NO). NO produced from eNOS is a potent vasodilator, anti-aggregatory autacoid important in blood pressure control, vascular remodeling and limiting the progression of atherosclerosis. Our work has focused on understanding the basic cell biology of eNOS as it pertains to the regulation of NO production. eNOS is co-translationally N-myristoylated (at glycine-2) and post-translationally cysteine palmitoylated (at cysteines 15 and 26). These lipid modifications are important for eNOS trafficking into the Golgi region and into cholesterol and glycolipid rich microdomains of the plasma membrane. Mutations of either site influence eNOS trafficking and block stimulated NO release without influencing eNOS activity in broken cell lysates (1-3). These data suggest that impairment in the trafficking of eNOS can influence the ability of the endothelium to produce NO. Using eNOS tagged with the green fluorescent protein (eNOS-GFP), we demonstrated that the first 35 amino acids in conjunction with the lipid modifications is sufficient for Golgi targeting (4). More recently it is clear that eNOS moves in a dynamic manner between the Golgi complex and cholesterol rich plasmalemma domains called caveola. Data will be presented demonstrating that eNOS-GFP moves depending on the state of endothelial cell confluency and that the different pools of the enzyme move at different rates using GFP photobleaching and imaging techniques.

Since the compartmentalization of eNOS is critical for optimal NO production, we hypothesized that proper subcellular targeting of the enzyme places eNOS is an environment containing NOS regulatory proteins. Indeed, eNOS interacts with many different proteins based on metabolic labeling experiments including a 22 kDa protein identified as caveolin-1 (CAV-1) (1,5,6). Caveolin is an integral membrane protein and the primary coat protein of the caveolae. eNOS binds to caveolin in endothelial cells and directly in vitro through a particular region of caveolin called the "scaffolding domain". This domain (amino acids 82-101 in caveolin-1) also serves as a docking site for other signaling molecules such src related tyrosine kinases, certain heterotrimeric G-protein subunits and the small G protein Ras. Work form our lab has identified a site on eNOS that serves as a binding site for caveolin termed the caveolin-binding domain (CBD). Caveolin or peptides derived from the scaffolding domain of caveolin can inhibit eNOS directly and reduce electron flux from the reductase to the oxygenase domain. Mutation of the putative binding site for caveolin on eNOS prevents the negative regulation by caveolin in co-transfection experiments. Thus, the identification of small molecules that disrupt the eNOS-caveolin regulatory cycle may have therapeutic relevance to improve endothelial dysfunction.

In addition to interactions with caveolin, we have identified heat shock protein-90 (Hsp90) as an eNOS regulatory protein (7). Hsp90 is an abundant cellular protein and is essential for signal transduction in yeast and in flies. Indeed, Hsp90 is required for the conformational maturation of src kinases and steroid hormone receptors and can bind to several protein kinases involved in signal transduction. Hsp90 associates with eNOS based on co-precipitation studies in human and bovine endothelial cells. Moreover, in transfection studies overexpression of Hsp90 enhances its binding to eNOS and results in increased eNOS activity in vitro; an effect that can be simulated with purified eNOS and Hsp90 in solution, suggesting that Hsp90 is a positive allosteric activator of eNOS. The physiological significance of this interaction is shown by the ability of agonists, namely vascular endothelial growth factor, histamine and fluid shear stress to rapidly initiate the recruitment of Hsp90 to the eNOS complex in a time frame consistent with the stimulated production of NO. Inhibition of Hsp90 signaling by ansamycins antibiotics geldanamycin or herbimycin A, attenuates agonist stimulated NO production and endothelium-dependent relaxation of isolated blood vessels. Thus, Hsp90 facilitates growth factor-, G protein- and mechano-signaling pathways that lead to the activation of eNOS. The relationships between caveolin and Hsp90 in the regulation of eNOS will be discussed.

1. Garcia-Cardena, G., Martasek, P., Masters, B. S., Skidd, P. M., Couet, J., Li, S., Lisanti, M. P., and Sessa, W. C. (1997) J Biol Chem 272(41), 25437-40

2. Liu, J., Garcia-Cardena, G., and Sessa, W. C. (1996) Biochemistry 35(41), 13277-13281

3. Liu, J., García-Cardeña, G., and Sessa, W. C. (1995) Biochemistry 34(38), 12333-40

4. Liu, J., Hughes, T. E., and Sessa, W. C. (1997) J. Cell Biol. 137, 1525-1535

5. Garcia-Cardena, G., Fan, R., Stern, D. F., Liu, J., and Sessa, W. C. (1996) J Biol Chem 271(44), 27237-27240

6. Ju, H., Zou, R., Venema, V. J., and Venema, R. C. (1997) J Biol Chem 272(30), 18522-5

7. Garcia-Cardena, G., Fan, R., Shah, V., Sorrentino, R., Cirino, G., Papapetropoulos, A., and Sessa, W. C. (1998) Nature 392, 821-824

The nitric oxide synthase (NOS) enzyme family consists of three major isoforms (1). The Ca²⁺-dependent brain enzyme (nNOS, type I) is mainly expressed in the central and peripheral nervous system but is also present in other tissues like skeletal muscle, macula densa, and placenta. In the brain, NO is formed in the course of excitatory neurotransmission via activation of nNOS by Ca²⁺ influx through the N-methyl-D-aspartate subtype of glutamate receptors. As a retrograde messenger, NO can produce a long-term potentiation of glutamate release from presynaptic nerve terminals in the hippocampus, a phenomenon which is thought to be involved in learning and memory formation. The endothelial NOS (eNOS, Type III), which is also Ca²⁺-dependent, is mainly found in vascular endothelial cells, where it is activated by hormones or in reponse to physical stimuli like blood flow and shear stress. The NO produced by the endothelium relaxes the vasculature and inhibits adhesion and aggregation of platelets. The inducible isoform of NOS (iNOS; type II) is is not constitutively expressed but synthesized de novo in a number of cell types, including macrophages, smooth muscle, cardiomyocytes, and microglia under inflammatory conditions. The most relevant triggers for iNOS expression are endotoxin and cytokines. Since the iNOS is Ca²⁺-independent it is permanently active and can act as high-output system generating substantial amounts of NO as required for the killing of bacteria, viruses and other pathogens. The overexpression of iNOS may contribute to the tissue injury and severe hypotension occurring in inflammatory disease states.

The NOS isozymes are fairly complex heme proteins containing two identical, functionally independent subunits with molecular masses of 130 kDa (eNOS and iNOS) or 160 kDa (nNOS). The human NOS genes are located on chromosomes 12 (nNOS: 150 kb, 29 exons), 7 (eNOS: 21-22 kb, 26 exons), and 17 (iNOS: 37 kb, 26 exons) (2). As may be expected from the complexity and structural diversity of the NOS genes, there are several reports on alternatively spliced variants. For instance, two catalytically active splice forms of nNOS (nNOS ß and [gamma]) have been identified in mice with targeted deletions of exon 2. These splice forms might be important in certain brain regions and probably account for the residual nNOS activity in nNOS knockout mice.

Each NOS isoform has the same layout of catalytic domains (3, 4) : a carboxy-terminal reductase with one binding site each for FAD, FMN and NADPH, and an amino-terminal oxygenase section. The crystal structure of the oxygenase half of iNOS was recently reported (5). Calmodulin (CaM), which binds at the amino-terminal side of the reductase is essential for the electron flow from the reductase to the oxygenase domain. An important difference between the isoenzymes is that, while in eNOS and nNOS, CaM binding and activation respond to physiological changes in Ca²⁺, iNOS already binds CaM and is fully active at the lowest Ca²⁺ concentrations encountered in vivo (6, 7) . The oxygenase domain contains bound heme and the binding site for the pteridine cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (H₄biopterin). The role of H₄biopterin in NOS catalysis is not well understood. As an allosteric effector, the pterin apparently stabilizes the protein in its dimeric state and maintains the heme in the catalytically active high-spin state (8, 9) . However, those allosteric effects are also produced by high affinity binding of the functionally inactive 4-amino analog of H₄biopterin (10, 11) . This hints strongly that the conformational effects of H₄biopterin may not alone be sufficient to activate the enzyme, and that the cofactor may be a direct chemical participant at some point in the reaction. Based on recent functional and spectroscopic data obtained at low-temperature (-15 to -30 deg.C) we have proposed that H₄biopterin is essential for the reductive activation of the oxy-ferri-heme complex resulting in formation of the perferryl species that eventually carries out the oxidation of the substrate. This hypothesis agrees well with our previous findings that binding of H₄biopterin is required to prevent the uncoupling of reductive oxygen activation that results in the formation of superoxide (12). Also, the hypothesis explains why the tetrahydro but not the oxidized dihydro forms of pterins are active cofactors (13).

The binding of H₄biopterin to NOS shows strong negative cooperativity between the two binding sites of the homodimer, which are located one on each of the two subunits (9). The first molecule of H₄biopterin binds with a dissociation constant in the low nanomolar range, while the second dissociation constant is about three orders of magnitude larger. This explains the observation that the enzyme as purified usually contains one molecule of H₄biopterin per dimer. The H₄biopterin-free and H₄biopterin-containing subunits appear to be catalytically independent (14). This seems likely to have important physiological implications. Over a wide range of H₄biopterin concentrations, one site per dimer will be occupied, so that the overall rate of NO synthesis could be fairly insensitive to fluctuations in H₄biopterin concentration. However, the activity of the H₄biopterin-free subunit should be remembered, in light of its ability to catalyze O₂^- production. O₂^- reacts rapidly with NO to form peroxynitrite, which is cytotoxic. Due to anticooperative H₄biopterin binding to the two subunits of homodimeric nNOS, the enzyme normally generates stoichiometric amounts of NO and O₂^- which combine rapidly to form the potent oxidant peroxynitrite (ONOO^-) (15).

Under physiological conditions cells appear to be protected from peroxynitrite injury by the synergistic action of superoxide dismutase (SOD) and reduced glutathione (GSH). While removal of superoxide by SOD shifts the L-arginine/NO pathway towards free NO, a not well characterized reaction of NO/O₂^- with GSH at physiological concentrations results in formation of S-nitrosoglutathione (GSNO) (16). Unlike free NO, GSNO is fairly stable in the presence of O₂ and may serve as transport or storage form of NO. The homolytic cleavage of GSNO, resulting in release of free NO and formation of GSSG, is catalyzed by reduced trace metal ions like Cu⁺, but enzymatic mechanisms have also been described (17, 18) .

It is not clear whether thiol nitrosation by NOS-derived NO/O₂^- is important for normal cell function or plays a role rather in the cellular defense against oxidative injury. Significantly reduced cellular levels of GSH and SOD are found in oxidative stress-related diseases (19). Therefore, the possibility that nNOS could work effectively as a peroxynitrite synthase under such conditions might account for the apparent contribution of this isoenzyme to brain ischaemia-reperfusion injury and other neurodegenerative disorders (20-23).

2. Förstermann, U. and Kleinert, H. (1995) Naunyn-Schmiedeberg's Arch. Pharmacol. 352, 351-364

3. Hemmens, B. and Mayer, B. (1997) in Methods in Molecular Biology (Titheradge, M.A., eds) pp. 1-32, Humana Press Inc., Totowa, N.J.

5. Crane, B.R., Arvai, A.S., Ghosh, D.K., Wu, C.Q., Getzoff, E.D., Stuehr, D.J., and Tainer, J.A. (1998) Science 279, 2121-2126

6. Cho, H.J., Xie, Q.W., Calaycay, J., Mumford, R.A., Swiderek, K.M., Lee, T.D., and Nathan, C. (1992) J. Exp. Med. 176, 599-604

7. Salerno, J.C., Harris, D.E., Irizarry, K., Patel, B., Morales, A.J., Smith, S.M.E., Martasek, P., Roman, L.J., Masters, B.S.S., Jones, C.L., Weissman, B.A., Lane, P., Liu, Q., and Gross, S.S. (1997) J. Biol. Chem. 272, 29769-29777

8. Klatt, P., Schmidt, K., Lehner, D., Glatter, O., Bächinger, H.P., and Mayer, B. (1995) EMBO J. 14, 3687-3695

9. Gorren, A.C.F., List, B.M., Schrammel, A., Pitters, E., Hemmens, B., Werner, E.R., Schmidt, K., and Mayer, B. (1996) Biochemistry 35, 16735-16745

10. Mayer, B., Wu, C., Gorren, A.C.F., Pfeiffer, S., Schmidt, K., Clark, P., Stuehr, D.J., and Werner, E.R. (1997) Biochemistry 36, 8422-8427

11. Pfeiffer, S., Gorren, A.C.F., Pitters, E., Schmidt, K., Werner, E.R., and Mayer, B. (1997) Biochem. J. 328, 349-352

12. Heinzel, B., John, M., Klatt, P., Böhme, E., and Mayer, B. (1992) Biochem. J. 281, 627-630

13. Klatt, P., Schmid, M., Leopold, E., Schmidt, K., Werner, E.R., and Mayer, B. (1994) J. Biol. Chem. 269, 13861-13866

14. Gorren, A.C.F., Schrammel, A., Schmidt, K., and Mayer, B. (1997) Biochemistry 36, 4360-4366

15. Beckman, J.S. and Koppenol, W.H. (1996) Am. J. Physiol. - Cell Physiol. 40, C1424-C1437

16. Mayer, B., Pfeiffer, S., Schrammel, A., Schmidt, K., Koesling, D., and Brunner, F. (1998) J. Biol. Chem. 273, 3264-3270

17. Gorren, A.C.F., Schrammel, A., Schmidt, K., and Mayer, B. (1996) Arch. Biochem. Biophys. 330, 219-228

18. Gordge, M.P., Hothersall, J.S., Neild, G.H., and Dutra, A.A.N. (1996) Br. J. Pharmacol. 119, 533-538

19. Winterbourn, C.C. and Munday, R. (1990) Free Radical Biol. Med. 8, 287-293

20. Huang, Z.H., Huang, P.L., Panahian, N., Dalkara, T., Fishman, M.C., and Moskowitz, M.A. (1994) Science 265, 1883-1885

21. Dorheim, M.A., Tracey, W.R., Pollock, J.S., and Grammas, P. (1994) Biochem. Biophys. Res. Commun. 205, 659-665

22. Schulz, J.B., Matthews, R.T., Muqit, M.M.K., Browne, S.E., and Beal, M.F. (1995) J. Neurochem. 64, 936-939

23. Ovadia, H., Rosenmann, H., Shezen, E., Halimi, M., Ofran, I., and Gabizon, R. (1996) J. Biol. Chem. 271, 16856-16861

A principle role of the endothelium, in addition to its other vascular homeostatic functions is the regulation of vascular tone. Physical stimuli such as fluid shear stress and pulsatile stretch are sensed by the endothelium and lead to an enhanced synthesis and release of endothelium-derived vasoactive autacoids, the most important of which is nitric oxide (NO). The constitutively expressed NO synthase (NOS) isoform present in endothelial cells (eNOS) binds calmodulin in a Ca2+-dependent manner, and therefore can be activated by agonists which increase [Ca2+]i. Earlier studies using a crude eNOS preparation from native endothelial cells however demonstrated that enzyme activity can be observed even at Ca2+ concentrations as low as 10 nM indicating that NO may also be formed via a Ca2+-independent pathway. Little physiological relevance was attributed to this phenomenon and the identification of a calmodulin-binding domain in the primary structure of the endothelial NO synthase together with the finding that calmodulin-binding proteins inhibited enzyme activity strengthened the hypothesis that the binding of a Ca2+/calmodulin complex is essential to activate the constitutive enzyme. There is now convincing experimental evidence showing that eNOS may be stimulated by two independent signalling pathways and is differentially activated by receptor-dependent agonists and mechanical stimuli [1]. Much of the available information relating to shear or stretch-induced signalling pathways and NO production have been obtained in models in which endothelial cells, cultured under static conditions, are exposed to acute increases in either shear stress or cyclic strain. Although the results of such experiments are informative, the situation in native endothelial cells is markedly different since these cells are continuously exposed to fluctuating levels of shear stress and pulsatile stretch. Indeed, some intracellular events such as the increase in [Ca2+]i in cultured endothelial cells tend to be transient and are unlikely to be representative of the real shear stress-induced response in vivo.

A number of intracellular signal transduction pathways are initiated by acute increases in fluid shear stress in cultured endothelial cells and include the activation of phospholipase C and a rapid increase in intracellular levels of inositol-1,4,5-trisphosphate, activation of a Ca2+-independent isoform of protein kinase C, as well as an elevated production of prostacyclin and free radicals. The phosphorylation of small heat shock proteins and the induction of some early response genes can also be detected shortly after application of shear stress (c-myc after several minutes, c-fos, c-jun within 2 hrs), as well as activation of the transcription factors AP-1 and NF-kB (for review see [2]).

While the activation of eNOS in response to Ca2+-elevating, receptor-dependent agonists, such as acetylcholine and bradykinin is relatively transient, the activation of eNOS following the application of fluid shear stress differs in that it can be maintained for several hours (i.e., as long as shear stress is applied). This shear stress-induced NO production is insensitive to the removal of extracellular Ca2+ and is not inhibited by the calmodulin antagonist, calmidazolium which abrogates the agonist-induced vasodilatation to acetylcholine [3,4]. This apparently Ca2+-independent eNOS activation is inhibited by the tyrosine kinase inhibitors erbstatin A and herbimycin A suggesting that the tyrosine phosphorylation of eNOS or an associated regulatory protein is crucial for its Ca2+-independent activation. To determine the effects of shear stress on the phosphorylation of eNOS we incubated porcine aortic endothelial cells with 32P for 6 hrs prior to cell stimulation, then immunoprecipitated eNOS and subjected the hydrolysed protein to two dimensional phosphoamino acid analysis. In unstimulated 32P-labelled endothelial cells eNOS was phosphorylated on serine, threonine and tyrosine residues. Fluid shear stress (2-5 min) rapidly increased the phosphorylation of eNOS on serine and tyrosine residues (P-Tyr by 267?44%; P-Ser by 130?20%). Phosphorylation on P-Tyr and P-Ser returned to basal levels within 30 min and decreased below basal upon prolonged exposure to shear stress (up to 4 hrs). eNOS phosphorylation on tyrosine, but not serine residues, was prevented by herbimycin A. Removal of extracellular Ca2+ on the other hand prevented the shear stress-induced phosphorylation of eNOS. Thus the initial transient phosphorylation of eNOS in response to fluid shear stress appears to be a Ca2+-dependent phenomenon. As an enhanced tyrosine phosphorylation of eNOS was therefore unlikely to regulate the Ca2+-independent activation of eNOS we investigated other shear stress-induced changes in eNOS. The most marked effect of shear stress on the eNOS protein was its redistribution from a detergent (Triton X-100)-soluble to an insoluble cell fraction. A change in eNOS detergent solubility was first evident 15 to 30 min after initiating fluid shear stress and was sensitive to the tyrosine kinase inhibitors erbstatin A and herbimycin A as well as the chaperone-binding agent geldanamycin. A pharmacologically identical (Ca2+-independent, tyrosine kinase inhibitor-sensitive) activation of eNOS and alteration in its detergent solubility can be induced by protein tyrosine phosphatase inhibitors underlining the importance of an increase in the tyrosine phosphorylation of endothelial proteins in the sustained activation of eNOS. However given that the maintained activation of eNOS was not associated with the hyperphosphorylation of eNOS, the phosphorylation/activation of an eNOS-associated regulatory protein, rather than eNOS itself, appears to be crucial for its Ca2+-independent activation

[5]. A change in the detergent solubility of a protein is frequently indicative of the formation of a protein-protein or protein-lipid complex for example, p91-phox, p22-phox, p47-phox and p67-phox in activated neutrophils; focal adhesion kinase (pp125FAK) and Crk-associated substrate (p130Cas) in fibroblasts. In both native and cultured endothelial cells a number of proteins are specifically co-precipitated with eNOS, most notably proteins corresponding to ~200, 90, 69-72 and 53 kDa. The Ca2+-elevating receptor-dependent agonist bradykinin, which does not alter the detergent solubility of eNOS, did not change the pattern of eNOS associated proteins. In contrast, the tyrosine phosphatase inhibitor, phenylarsine oxide which Ca2+-independently activates eNOS and renders it Triton-insoluble, decreased the recovery of 210 and 69-72 kDa proteins from the detergent-soluble fraction. Proteins of ~103 and 87-91 kDa were recovered from the soluble fraction of cells treated with phenylarsine oxide but not with solvent, bradykinin or the combination of herbimycin A and phenylarsine oxide. These observations suggest that eNOS exists as part of a multi-molecular complex and that the Ca2+-independent activation induced by phenylarsine oxide seems to be linked to changes in the constituents of this complex and results in the alteration of its detergent solubility.

Moreover since these changes were sensitive to herbimycin A, this process appears to involve activation of tyrosine kinases.

Apart from fluid shear stress hemodynamically relevant cell-cell-generated forces affect endothelial NO production. One example is the isometric contraction in which the development of contractile force within the smooth muscle cell layer counteracts the distending transmural pressure. Under such conditions there is a relative displacement of opposing cell layers within the vascular wall (e.g., smooth muscle cells vs. elastic lamina and endothelial cells) despite the fact that no net movement occurs. Although the displacement induced may be subtle, the close physical arrangement of endothelial focal adhesion contacts and the smooth muscle would tend to suggest that the forces developed at the abluminal surface of endothelial cells may be greater than those generated by shear stress on the luminal surface. While these forces cannot be expressed as a simple physical term, isometric contraction of endothelium-intact arterial segments has been demonstrated to elevate NO production. In rings preconstricted under isometric conditions with PGF2a, up to 40% of the maximal phenylephrine-induced contraction, the NOS inhibitor, NGnitro-L-arginine, elicited an additional contraction which was dependent on the level of preconstriction. A less pronounced effect of NGnitro-L-arginine was observed in rings preconstricted to over 50% of the maximally inducible tone. These observations indicate that stretch elicited by isometric contraction, within a certain range, activates eNOS in endothelial cells.

More direct evidence for the release of NO by isometric contraction was obtained in bioassay experiments where the superfusate from isometrically contracted rings increased cyclic GMP levels in detector segments. This NO production and the supplementary NGnitro-L-arginine-induced increase in vascular tone were inhibited by the non-selective kinase inhibitor staurosporine and the tyrosine kinase inhibitors erbstatin A and herbimycin A, whereas the calmodulin antagonist calmidazolium and the selective protein kinase C inhibitor Ro 31-8220 were without effect. Coincident with the NO formation was an increase in endothelial tyrosine phosphorylation which also correlated with the preconstriction level. Thus isometric contraction, tyrosine phosphatase inhibitors and fluid shear stress appear to activate the Ca2+/calmodulin-independent formation of NO via a similar tyrosine kinase-linked signalling pathway.

1. Fleming I, Bauersachs J, Busse R: Calcium-dependent and -independent activation of the endothelial NO synthase. J.Vasc.Res. 1997;34:165-174

2. Busse R, Fleming I: Pulsatile stretch and shear: physical stimuli determining the production of endothelium-derived relaxing factors. J.Vasc.Res. 1998;35:73-84

3. Ayajiki K, Kindermann M, Hecker M, Fleming I, Busse R: Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells. Circ.Res. 1996;78:750-758

4. Kuchan MJ, Frangos JA: Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am.J.Physiol. 1994;266:C628-C636

5. Fleming I, Bauersachs J, Fisslthaler B, Busse R: Calcium-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress. Circ.Res. 1998;81:686-695

Nitric oxide synthases (NOS) catalyze the NADPH and O₂-dependent conversion of L-arginine to L-citrulline and various N-oxides including NO, ONOO^- and possibly others. Whilst many of the biochemical features of NO synthases parallel the cytochrome P₄₅₀/NADPH-dependent cytochrome P₄₅₀ reductase enzyme system, an important difference is the dependence of all NOS for maximal activity on the pteridine (6R)-5,6,7,8-tetrahydro-L-biopterin (H₄Bip). The reason for this is unclear. Basal NOS activity has been attributed to enzyme-associated pterin. Pteridines and folates are widespread cofactors with diverse functions both in mammalian cells and lower life forms. Whereas mammals can synthesis pterins, folates are essential and have been clinically targeted with the development of anti-folates as chemotherapeutic and cytostatic agents. To date, there has been no systematic approach to interfere with the function of pteridines, e.g. H₄Bip. Interestingly, the NOS pterin binding site appears to be distinct from that of aromatic amino acid hydroxylases, a large family of H₄Bip-requiring enzymes involved in neurotransmitter biosynthesis. Thus, pterin-based inhibitors and activators of NOS are both interesting biochemical tools to elucidate the function of H₄Bip in NO synthesis and potential drug candidates to modulate NO synthesis in intact cells or in vivo. A new class of pharmacologically active compounds, termed anti-pterins, displaced endogenous H₄Bip, but surprisingly without affecting basal activity. Moreover, pterin content of purified NOS was frequently lower than basal activity, and pterin-free NOS could not be saturated with exogenous H₄Bip. These findings suggest that basal NOS activity may be independent of H₄Bip. Instead, H₄Bip binds to the oxygenase/dimerization domain, prevents monomerization during L-arginine turnover and stabilizes NOS activity.

Some of the earliest inhibitors of NOS were derived from the substrate L-arginine, e.g. the guanidine and amidine derivatives, N^[omega]-methyl-L-arginine, N^[omega]-nitro-L-arginine, its methyl ester and the N^{-iminoethyl-L-ornithine.}Although the co-product of NOS L-citrulline does not feed back on NOS, analogs of L-citrulline such as L-thiocitrulline, L-homothiocitrulline and S-methyl-L-thiocitrulline, which all contain replacements of the thioureido oxygen by sulphur, are potent inhibitors of the enzyme. The fact that the guanidino moiety of L-arginine is the natural substrate of NOS has led to the development of cyclic and acyclic amidines, guanidines and isothioureas as active site inhibitors with reversible and irreversible modes of action. It is a short conceptual step from a cyclic amidine to pyridyl precursors like 2-aminopyridines, and from there to aniline derivatives which display some isoform selectivity but are less potent. Only few of the inhibitors that have been published appear to be acting at one of the other cofactor sites of NOS. Whereas diphenyleneiodonium is known to be the most prominent flavin cofactor binding site inhibitor also some quinazoline or aromatic hydroxamic acid derivatives act at the flavin or H₄Bip cofactor site, respectively. Furthermore, some indazoles or imidazoles bind as heme cofactor ligands and thereby inhibit NOS activity. Thus, we developed a series of novel H₄Bip derivatives including also 4-amino substituted compounds. However, many of these compounds lack isoform selectivity and appear to interact with other binding sites of NOS. To establish a structure activity relationship of the NOS-I pterin/anti-pterin binding site the functionalities in 2, 4, 5, 6 and 7-position were systematically varied. Increasing the basicity of the 4-amino function of the aromatic 4-amino pterin derivative resulted in potent and complete inhibitors of NOS-I activity with IC₅₀-values in the low micromolar range. This inhibition is consistent with an optimized hydrophilic interaction with hydrogen providing groups of the NOS pterin binding site. This notion agrees with the recently published crystal structure of the dimeric murine iNOS oxygenase domain (NOS_ox). In contrast, varying the substitution pattern in the 2, 5 and 7-position had no significant influence on the inhibition of NOS-I activity. In contrast, bulky substituents in 6-position like phenyl- or mesityl groups markedly increases the inhibitory potency of these possibly as a consequence of a steric interaction with the NOS-I enzyme.

Methods--NOS-I (nNOS) was isolated from porcine brain cerebellum or Sf9 cells expressing recombinant human NOS-I by ammonium sulphate precipitation and 2',5'-ADP-sepharose affinity chromatography and eluted with excess 2'-AMP as previously described. To exclude the possibility that this purification method influenced the inhibitory profile of the anti-pterins, NOS-I was additionally purified by DEAE ion exchange chromatography instead of ammonium sulphate precipitation. NOS-I was further purified to homogeneity by using calmodulin-sepharose 4 B affinity chromatography. Enzyme activity was determined by the calcium/calmodulin-dependent conversion of ³H-L-arginine to ³H-L-citrulline either in the absence (basal activity) or presence of 2 uM H₄Bip (maximal activity). The dehydration of 6-methylcarbinolamine was followed spectrophotometrically at 245 nm in 10 mM Tris/HCl (pH 7.4). The influence of the anti-pterins on NOS-I activity was screened initially at an inhibitor concentration of 100 uM. Subsequently, concentration response curves were constructed for the most effective inhibitors and the corresponding IC₅₀-values determined by non-linear regression analysis using the UltraFit computer software (Biosoft; Cambridge, U.K.). To determine enzyme-bound pterin, NOS was oxidized with 0.2 M I₂ and 0.5 M KI in the presence of either 0.5 M HCl (acidic oxidation) or 0.1 M NaOH (alkaline oxidation) and kept for 1 h in the dark at room temperature. The quantitative difference between pterin recovery under acidic and alkaline condition is a measure of H₄Bip. Samples were analyzed by reverse-phase HPLC using a LiChroCart 250/4 Purospher RP 18 (5 um) column (Merck, Darmstadt, Germany) with fluorometric detection. To determine the effect of anti-pterins on NOS activity in intact cells, N₁E-115 cells, a murine neuroblastoma cell line expressing NOS-I, were incubated in Krebs Ringer buffer. NO formation and release was quantified spectrophotometrically as NO₂^- using the Griess reaction. In enzyme structural studies, the monomer/dimer ratio of NOS-I was determined by FPLC gel filtration chromatography using a Superose 6 HR 10/30 column (Pharmacia Biotech, Freiburg, Germany) at 4 deg.C; 20 mM TEA, pH 7.5, 150 mM NaCl, 5% ethylene glycol.

Possible Chemical Interactions Within the H₄Bip Binding Pocket--4-Amino substitution of H₄Bip yields a potent inhibitor of NOS (Werner et al. 1996, Biochem. J. 320, 193). In order to establish a structure-activity relationship (SAR) of the NOS pterin/antipterin binding site, the pterin substitution pattern and functionalities, of the 4-amino pterin derivative, was systematically varied. This yielded several new, potent derivatives as inhibitors of NOS-I activity and a further insight into the NOS pterin binding pocket and an adjacent exosite. Varying the substitution pattern in the 2 position led to no significant change in the inhibitory potential of the aromatic 4-amino derivatives suggesting that this functionality is not important in the activation of NOS-I enzyme. Similar conclusions can be drawn for the 7 position, since chemical modifications of this position alone also showed no influence on the inhibitor profile of these anti pterins. This interpretation agrees well with the recently published crystal structure of the dimeric murine iNOS oxygenase domain containing bound H₄Bip (Crane et al. 1998, Science 279, 2121). In this study, a hydrophilic interaction in the catalytic center between the 4-keto function of the natural H₄Bip with the propionic-acid side chain of heme and the free amino group of the guanidine rest of Arg³⁷⁵ was observed. Given the high degree of sequence homology, one would expect that these interactions would also be applicable to native NOS and anti-pterins. One would predict that by increasing the basicity of the 4 position and therefore the ability to form a more effective bonding interactions with the hydrogen donating heme and structural arginine, inhibitor potency should increase. This is the case for the 4 keto substitution of the natural cofactor through an aromatic 4 amino derivative. In agreement, 4 amino dialkylation produced NOS inhibitors with greater potency. The decrease of the pK_b-values of their free bases correlated with their IC₅₀ values. Furthermore, Crane et al. demonstrated a hydrophilic interaction between the 9,10-dihydroxylated propyl side chain of the natural H₄Bip and the phenylalanine (Phe⁴⁷⁰) and serine rest (Ser¹¹²) of the enzyme, this being preferred as they are located spatially close together. In contrast, this chemical interaction appeared to be functionally insignificant for the 6 position of the aromatic pterin derivatives. This difference may be due to the planar nature of the pyrazin ring of the aromatic pterin derivatives which probably fails to effectively insert in the NOS pterin binding pocket, although an increased [pi]-electron stacking with Trp⁴⁵⁷ should be favoured when compared to conformations of the 6-substituted 4-amino- and 5,6,7,8-tetrahydro-pterin derivatives. The additional inhibitory influence of the 6 position of the aromatic compounds seem to be rather due to hydrophobic interactions with the aromatic part of Phe⁴⁷⁰. Interestingly, substitution of the 6 position with either a phenyl- or mesityl group of the aromatic 4 amino pterin derivative alone had no effect; but combined with functionalities that increase the basicity of the 4 position, especially with a mesityl group in 6 position, resulted in potent inhibitors of NOS-I. The apparent lack of potency of the aromatic 6-phenyl versus 6-mesityl substituted 4-amino-pterines may be related to (a) lack of positive inducible effects that could be transferred over the aromatic ring system to the 4-N position and thereby further increase basicity, (b) an enlarged spatial demand and steric repulsion and as a consequencec of this supports the 4 amino pterin derivative binding into the possible 4-position binding pocket of NOS, (c) lack of H-bond formation between the 6-MeO-group and Ser¹¹². This could be the driving force for the stronger binding of either the 4-dialkylated 6-mesityl and morpholine-substitued compounds to NOS, whereas the possible counteractive additional hydrophilic NMe-interaction, in case of the piperazine ring substituted compounds, could not be compensated by this effect. In the case of the 4-piperidine substituted compounds, this additional hydrophilic interaction due to a -CH₂ at the 4 position of the heterocyclic ring system is not possible which is why the inhibitory potential through a mesityl group in 6 position could not be observed.

In conclusion the inhibitory effect of the aromatic 4-amino substituted anti pterins appears due to an increased hydrophilic interaction of the 4 position within the binding pocket of NOS compared to the naturally occuring (6R)5,6,7,8-tetrahydro-L-biopterin cofactor, and, on the other hand, due to a hydrophobic interaction of the bulky aromatic substitutents in the 6 position of the aromatic 4 amino pterin derivatives with Phe⁴⁷⁰ from NOS. An intimate localization of the pterin binding pocket adjacent to the heme and arginine substrate binding domain in the crystal structure of iNOS_ox are supported by our findings using the 4 amino pterin derivatives and hypothesis of binding site interactions based on their structural changes. From our structure-activity relationship we suggest that the upper portion of the pterin molecule (positions 4,5,6 of the H₄Bip cofactor) is responsible for efficient insertion and binding of H₄Bip into the binding pocket of NOS. Several of these observations may be highly species- and isoform specific. We found that the inhibitory effect of selected compounds on human and NOS I-III isoforms activity was slightly lower than as it was in the case of the native porcine NOS I, or in some cases absent. Thus, there is great potential for isozyme-selective inhibitors of NOSs by following the ant-pterin approach.

Activating and Stabilizing Effects of H₄Bip--Further enzyme kinetic studies using porcine cerebellum NOS-I revealed two main, distinct inhibitor profiles. Type I anti-pterins inhibited exclusively the H₄Bip-stimulated NOS activity without inhibiting basal NOS activity, i.e. that activity which is observed in the absence of exogenous H₄Bip. Hitherto, basal NOS activity had been generally accepted to be due to tightly bound, endogenous H₄Bip which co-purifies with NOS and cannot be readily displaced. Surprisingly, the type I anti-pterin PHS-32 under different conditions effectively displaced endogenous pterin (> 80%) without any effect on basal enzyme activity. The most likely explanation for this finding is that the basal activity of NOS is H₄Bip-independent. The possibility that PHS-32 not only displaced H₄Bip but also acted as partial agonist would equally exclude a catalytic mechanism for NOS-associated pterin. Three additional lines of evidences further corroborated that basal NOS activity may have no absolute requirement for H₄Bip. In several native and recombinant NOS-I preparations, no correlation between the endogenous H₄Bip content (% of NOS monomers) and basal citrulline accumulation (% of V_max) was found. We propose that purified NOS-I is comprised of two conformational states. One is responsible for a H₄Bip-independent, basally active enzyme (NOS*). The remaining enzyme (NOSdeg.) is inactive or immediately inactivates without the addition of H₄Bip. In a subsequent series of preincubation experiments, we observed that NOSdeg. cannot be converted into NOS* by a short exposure to H₄Bip prior to the activity assay. This excluded again the possibility that binding of H₄Bip alone modifies the enzyme and is sufficient to induce basal activity or NOS*. NOSdeg. is not a purification artifact and is physiologically co-expressed with NOS*, since NOSdeg.-specific anti-pterins like PHS-32 were are also effective inhibitors of NO synthesis in intact cells. The cellular regulation of each NOS isoform into NOS* and NOSdeg. may be an important mechanism of physiologically controlling NOS activity and may explain some clinical observations. For example, in some patients with phenyl ketonuria, the cause of the disease is due to their impaired ability to synthesizes H₄Bip. However, as these patients do not show clinical signs that might be expected if H₄Bip deficiency led to decreased NOS activity with consequent impairment in immune function, blood pressure and neuronal regulation, NOS in these patients might be at least partly independent of H₄Bip. Finally, in all H₄Bip-utilizing enzymes, the intermediate 4a-hydroxy-pterin is converted to the quinoid-H₂Bip. This conversion occurs spontaneously (with some side-reactions to 7,8-H₂Bip) and is augmented by PCD. With respect to NOS-I, PCD does neither increase the basal NOS activity, the V_max nor the affinity for H₄Bip. Moreover, NOS-I does not contain PCD activity itself.

Our data collectively suggest that endogenous H₄Bip alone is not sufficient to account for basal NOS activity. When we investigated the effect of H₄Bip on the native quaternary protein structure of catalytically active NOS-I, an essential stabilizing function of H₄Bip was revealed. While purified NOS-I exists as a stable homodimer and monomerization was only described to occur under denaturing conditions, i.e. in the presence of 4% SDS or 5 M urea, we observed under physiological conditions, i.e. during catalytic L-arginine turnover, that NOS-I rapidly dissociated in a time-dependent manner into inactive monomers correlating with a dramatic loss of activity after 3-4 min of incubation. The addition of H₄Bip prevented both the dissociation into inactive monomers as well as the corresponding loss in activity during turnover. Although dimer stabilization may not explain all of the actions of H₄Bip, it may contribute to the prevention of activity loss after several rounds of L-arginine turnover and it is conceivable that H₄Bip is not essential from the start of catalysis. A stabilizing effect of H₄Bip is further supported by a recent report showing that H₄Bip prevented monomerization of the NOS-II_ox domain. The dependence of NOS* on H₄Bip after 3-4 min of incubation may explain why expression of NOS under pterin-free conditions has resulted in instable enzymes with reduced activity requiring H₄Bip from the start. Most likely, the expression of NOS under these conditions predominantly yields an inactive NOS. In agreement with a stabilizing effect of H₄Bip within the catalytic center of NOS we localized the NOS-I pterin/anti-pterin binding site to a 341 amino acid sequence of the NOS_ox domain using a [³H]labeled type II anti-pterin as a photoaffinity probe. Interestingly, the N-terminus of NOS-I, specific for this isoform, appears to modulate pterin binding. The inclusion of heme in this fragment agrees with an intimate H₄Bip:heme:arginine interaction. H₄Bip may act by preventing monomerization during L-arginine turnover without having an essential role for basal NOS activity. We are presently investigating whether NOS-derived products contribute directly to monomerization. Since anti-pterins were also effective in intact cells, they might become novel pharmalogical tools to downregulate pathological high NO-formation.

Cell death is defined by morphological criteria and is believed to occur by either necrosis or apoptosis. Necrotic death comprises cell and organelle swelling, ultimately followed by cell dissolution. In 1972, Kerr and coworkers defined the term "apoptosis". Apoptosis, a synonym for "programmed cell death" refers to the evolutionary conserved pathway of biochemical and molecular events leading to cell demise. Elements of a core program controlling the executive phase of apoptosis are expressed in virtually all cells. Apoptotic cells usually shrink and condense, display surface alterations, and cleave DNA into large and often small oligonucleosomal-sized (200 bp and multiples) fragments, while organelles and the plasma membrane retain their integrity. It is conceivable that apoptotic pathways converge to one, or very few, common final executive steps. These comprise the tumor suppressor p53, caspases (cysteine aspartase), or the regulatory role of Bcl-2 family members.

NO^,generated at a high quantity by activated macrophages is part of the inflammatory response against bacteria, viruses, and tumor cells. In general, it is appreciated that a massive production of NO from L-arginine initiates cell injury. In several experimental systems such as macrophages NO-evoked death follows morphological and biochemical features that characterize apoptosis. Cell death is defined by upregulation of the tumor suppressor p53, activation of caspases, chromatin condensation, DNA fragmentation, and an altered expression of Bcl-2 family members. Apoptosis as a result of inducible NO-synthase activation is attenuated by NOS-inhibitors, thereby establishing a causative role of NO. However, experiments in p53 negative cells implied p53-independent pathways to be operative during NO-mediated apoptosis as well. In the meantime numerous reports confirmed the ability of NO to initiate apoptosis. This holds for macrophage cell lines, ß-cells (RINm5F), thymocytes, chondrocytes, mesangial cells, neurons, mast cells, vascular endothelial cells, smooth muscle cells, various tumor cells, and several more.

Overexpression of the antiapoptotic protein Bcl-2 rescued cells from apoptosis by blocking signal propagation downstream of p53 and upstream of caspases. As the biological milieu, which is balanced by internal and external stimuli, affects NO^-signaling via redox and additive chemistry, the toxicity of NO is not a constant value. As a result, cellular sensitivity towards NO varies and NO-transducing pathways may signal cell protection.

During our studies with rat mesangial cells we unexpectedly noticed that NO^-mediated apoptotic cell death was antagonized by the simultaneous formation of superoxide (O₂-). Part of the signal transmission of both, NO and O₂- may stem from their diffusion controlled interaction that results in the formation of peroxynitrite (ONOO-). We addressed the NO/O₂--interaction by exposing cells to NO donors and O₂- generating systems such as the redox cycler DMNQ (2,3-dimethoxy-1,4-naphtoquinone) or the hypoxanthine/xanthine oxidase system, thereby allowing a continuous radical formation. The balanced and simultaneous generation NO and O₂- turned out to be protective for mesangial cells, whereas the unopposed radical generation elicited apoptosis and in higher concentrations necrotic cell death. While apoptosis was accompanied by increased p53 and Bax expression, active caspases, and DNA fragmentation, these alterations were attenuated under conditions of NO/O₂--coadministration. Of particular importance is the simultaneous presence of both radicals. If the generation of either NO or O₂- is offset, protection is less efficient. We conclude that signaling mechanisms as a consequence of the NO/O₂--interaction redirect apoptotic initiating signals to harmless pathways. Although transducing mechanisms are uncharacterized so far, protection demands reduced glutathione (GSH). These studies are in analogy to in vitro experiments performed by Wink and colleagues. They observed GSSG formation by incubating NO donors, O₂-, and GSH and concluded NO-evoked nitrosative reactions to be quenched by the resultant oxidative stress. In some analogy NO attenuated O₂--mediated toxicity in chondrocytes or stretch-induced programmed myocyte cell death that resulted from O₂- formation, or abrogated toxicity of oxidized low-density lipoprotein in endothelial cells. Further, this may be in line with a protective function of NO during ischemia-reperfusion, peroxide-induced toxicity, lipid-peroxidation, or myocardial injury.

As a general concept it appears that in some systems the balanced formation and interaction of physiologically relevant radicals resembles a protective principle thereby eliminating harmful reaction that are operating as a consequence of unopposed radical generation.

Analyzing macrophage programmed cell death in detail, we realized that desensitization towards NO^-elicited apoptosis occurs upon preactivation with a combination of LPS and IFN- under conditions of blocked NOS or results form prestimulation with a low, nondestructive dose of NO donors. We determined induction of cyclooxygenase-2 (Cox-2) to represent a critical regulator of macrophage apoptosis. Non activated macrophages do not express Cox-2, whereas LPS/IFN-/NMMA caused protein expression within 6-12 h. In analogy, a nontoxic dose of GSNO promoted Cox-2 up-regulation and protection. A functional role of Cox-2 was assured in Cox-2 overexpressing macrophages. Protection was antagonized by the Cox-2 selective inhibitor NS-398 or by a stably transfected antisense Cox-2 expression vector. Obligatory for protection and/or expression of Cox-2 appears activation of the nuclear transcription factor NF-B (p50/p65-heterodimer formation). Degradation of I-B and activation of a luciferase reporter construct, containing four copies of the NF-B-site derived from the murine Cox-2 promoter confirmed NF-B activation. Furthermore, a NF-B decoy approach attenuated not only Cox-2 expression and inducible protection but also restored DNA fragmentation and p53 accumulation in response to high dose GSNO. These examinations provided evidence for an antiapoptotic role of NO, transmitted by NF-B activation. It is interesting to note that NF-B activation seems to represent a more general pathway to eliminate adverse, proapoptotic effects of diverse agonists. Besides, endorsed expression of heme oxygenase-1 (HO-1) or heat shock protein 70 also protects against NO damage. Consistent is the notion that cell survival demands the expression of protective proteins. It remains to be established how these protective proteins circumvent cell death by NO. Cellular protection as a result of NO formation is also noted in association with activation of soluble guanylyl cyclase or as a result of protein thiol modification, i.e. caspase inhibition.

The toxicity of NOis not a constant value. It is influenced by the existing biological milieu, i.e. relative rates of NO formation, oxidation and reduction, combination with oxygen, superoxide, and other biomolecules. In some systems activation of the iNOS generated sufficient amounts of NO to promote cell death that is defined by apoptotic features. However, not all systems that upregulate inducible-NOS enter the death pathway. Antagonistic and/or protective principles exist. Protection arises in the presence of a balanced rate of O₂--production which redirects cell destruction to cell protection. Besides adverse effects of NO the molecule also signals cell protection. NO-derived cell protection is rationalized by upregulating protective proteins such as heat shock proteins or Cox-2, by signaling pathways that demand cGMP formation, or thiol modification. It will be essential to define the versatility of NO-signaling mechanisms in relation to their apoptotic inducing ability. The switch from physiology to pathophysiology, the action of potentially protective and destructive NO-species, and the molecular recognition of these balances will be central to the understanding of the pro- and antiapoptotic actions of NO.

We thank the Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, and the European Community for support.

The process of carcinogenesis is currently viewed as a series of steps involving the inactivation of tumor suppressor genes and the activation of oncogenes. Both types of genetic change involve damage to DNA in the form of either mutations or deletions. In addition, the sequence of steps for transformation of a somatic cell requires alternation of DNA damage and cell division, resulting in expansion of the new target cell population.

The overall hypothesis to be presented in this lecture is that DNA damage, mutation, and cytotoxicity will arise as a result of nitrosative deamination, NO. radical reactions, and oxygen radical damage when target cells are exposed to generator cells that produce NO.. Depending upon the dose rate, total dose, types of cells, and other circumstances, NO. may drive cells into apoptosis through multiple pathways, or inhibit apoptosis and enhance mutation through damage to bases, strand breaks, and cross-links.

The chemistry of nitric oxide in oxygenated biological systems is extremely complex due to the large number of chemical species formed and the numerous parallel reactions to consider. The first pathway involves direct reaction of NO. with cellular targets after simple diffusion of NO..

Nitric oxide also reacts to form additional reactive species that can participate in other types of chemistry. One major fate of nitric oxide is reaction with superoxide anion (O2-.), to yield peroxynitrite, ONOO-. This is an extremely fast reaction due to the fact that both species are radicals. The rate of the nitric oxide/superoxide reaction is near the diffusion limit with a rate constant of 6.7 ' 109 M-1 s-1. This rate constant is approximately 3.5 times larger than that for the superoxide dismutase (SOD)-catalyzed decomposition of O2-. indicating that the nitric oxide/superoxide reaction may predominate over the superoxide/SOD reaction.

The formation of both nitric oxide and superoxide does indeed occur simultaneously in cells such as macrophages, neutrophils, Kupffer cells, and endothelial cells. In the vicinity of these cells, peroxynitrite may be present at high concentrations, although the mechanism and extent of ONOO- formation are strongly influenced by the relative fluxes of O2-. and NO. .

Nitric Oxide-Induced DNA Damage via the N2O3 Pathway. The formation of N2O3 can cause either direct or indirect damage. Direct damage results from the nitrosation of primary amines on DNA bases ultimately leading to deamination. Nitrosative deamination is also a well known consequences of the reaction of primary amines with acidic nitrite. In fact, the chemistry of N2O3 formation is nearly identical to that from nitrous acid, i.e. nitrite at an acidic pH, because it is the anhydride of HNO2. The overall reaction rates are actually higher based upon N2O3 at neutral or basic pH than at acidic pH because of the increased concentration of free amine under these conditions (1). There are a number of other NO[florin]-derived nitrosating agents that may be important under other conditions, however at physiological pH, N2O3 formation from nitric oxide has been demonstrated to be most important (2). Direct attack of N2O3 on DNA can lead to DNA deamination via diazonium ion formation (1). Hydrolysis of the diazonium ion completes the deamination. The end result of this process is the net replacement of an amino group by a hydroxyl group. Any DNA base containing an exocyclic amino group can undergo deamination upon reaction with N2O3.

Therefore, adenine, cytosine, 5-methylcytosine, and guanine can all be deaminated forming hypoxanthine, uracil, thymine and xanthine respectively. The potential consequences of these reactions vary from nucleoside to nucleoside. Deamination of guanine leads to xanthine formation. Mispairing of xanthine can cause a G:C->A:T transition, Xanthine is unstable in DNA and can depurinate readily leaving an abasic site, The cell may replicate past the abasic site following the "A" rule involving insertion of an adenine opposite the abasic site resulting in a G:C-> T:A transversion mutation (3). More likely, however, the abasic site may be cleaved by endonucleases resulting in the formation of single-strand breaks (4). Deamination of cytosine forms uracil which can give rise to a G:C-> A:T transaction mutation through mispairing. Given the high levels of uracil glycosylase in a cell, uracil can be easily repaired (5).

Methylation of cytosine to form 5-methylcytosine and subsequent deamination to thymine could also result n a G: C -> A: T transition. In addition, the deamination of adenine to hypoxanthine needs to be considered because mispairing of hypoxanthine with cytosine can lead to a A: T -> transition. Caulfield et al. (6), have studied the relative rates of NO[florin]-induced deamination of dG and dC in different environments in order to assess the importance of DNA structure in determining the reactivity of the different bases towards nitric oxide. Relative reactivity varied between deoxynucleosides, single and double stranded DNA. In all cases xanthine formation was twice that of uracil formation.

Recent evidence indicated that the reaction of deoxyguanidine with nitri oxide might actually be more complex than previously expected. Multiple products besides xanthine may be formed (7). Oxanosine, the ribonucleoside of this compound, had previously been isolated as a novel antibiotic in 1981 from a bacterial culture. The compound 2'-deoxyoxanosine was synthesized from oxanosine and exhibited a stronger antineoplastic activity than oxanosine. Whether 2'- deoxyoxanosine is actually formed from nitric oxide is somewhat questionable because in the studies carried out, the pH dropped to 2.9 and relatively high concentrations of nitrite were employed. NO[florin] treatment can also lead to the formation of single strand breaks in DNA. The formation of NO[florin]- induced strand breaks has been examined both in vitro and in vivo. Nguyen et al., demonstrated the NO[florin] causes both dose-and time-dependent DNA single-strand breaks in TK6 cells (8).

However, when supercoiled plasmid DNA was treated extracellularly with NO[florin] no nicking of DNA was observed (9). Surprisingly, when the same plasmid was transfected into CHO cells after treatment it was found to contain strand breaks. The most likely explanation for these results in that NO[florin] treatment can indirectly lead to single strand breaks via formation of xanthine which can depurinate leaving an abasic site. Intracellularly, these abasic sites are recognized by endonucleases which readily cleave them leading to formation of single strand breads. This hypothesis is supported by time course experiments measuring formation of abasic sites vs. strand breaks (9). CHO cells were treated with NO[florin] (85 nmol/ml min) for one hour. Directly after treatment only abasic sites were detected.

Abasic sites were reduced to background 12 h later but a high percentage of single-strand breaks were found suggesting that abasic sites may be cleaved to form strand breaks. DNA double-strand breaks were detected 24 h later indicating that unrepaired single-strand breaks may be converted to double-strand breaks with may be toxic to a cell. DNA intrastrand, DNA interstrand, and DNA-protein cross-link formation have been demonstrated upon treatment of DNA with nitrous acid. This raises the possibility that these products may be formed in NO[florin] -treated DNA via the intermediacy of N2O3. We have confirmed this possibility by quantitative analysis of the cross-links.

DNA Damage From Peroxynitrite. In contrast to nitric oxide, which is involved primarily in the deamination chemistry of DNA, most of the damage inflicted on DNA by peroxynitrite is oxidative in nature. DNA treatment with peroxynitrite generally leads to much more damage than treatment with an equivalent dose of nitric oxide. In addition to the higher levels of damage present in DNA after ONOO- treatment, the spectrum of damage also tends to be much more complex. All of this makes sense given that peroxynitrite is intrinsically much more reactive than nitric oxide.

Evidence that peroxynitrite is formed in vivo is steadily accumulating. As is the case with nitric oxide, peroxynitrite's transient nature required that its production in a cell be monitored indirectly. Beckman and co-workers were among the first to examine the formation of peroxynitrite from activated macrophages. They detected significant formation of peroxynitrite utilizing the nitration of 4-hydroxyphenylacetate as a marker of peroxynitrite activity. Lewis et al. (10), performed a kinetic analysis of the fate on NO[florin] synthesized by activated macrophages using endproduct measurements of nitrite and nitrate. Hydrolysis of N2O3 forms nitrite while ONOO- decay leads to nitrate formation. Their results indicate that approximately half of the released nitric oxide forms N2O3 while the remainder combines with O2 to form ONOO-. Interestingly, it appears from the Lewis study that ONOO- formation occurs partially extracellularly and not exclusively inside the macrophage. This can be inferred from the observation that adding SOD to the media significantly reduced ONOO- formation. If peroxynitrite formed only within the cell it should not have been affected by the extracellular addition of SOD. deRojas-Walker et al. (11), identified DNA deamination (xanthine) and oxidation (8-oxo-dG, FAPY-G, and 5-hydroxymethyl-uracil) base products in activated macrophage DNA . The formation of these products was inhibited by NG-methyl-L-arginine, a nitric oxide synthase inhibitor. Since both deamination and oxidation products were observed, their results demonstrate that effects of NO[florin] produced by a macrophage must be mediated not just by N2O3 but also by ONOO-. Immunohistochemical techniques using antibodies against 3-nitrotyrosine have also been used for ONOO- detection in animal models. For example, SJL mice, especially those bearing the RcsX tumor, suffer from chronic inflammation with activation of macrophages in the spleen and lymph nodes. Nitrotyrosine staining performed on SJL mice tissue sections showed positive results in cells adjacent to iNOS-expressing macrophages and also within the macrophages themselves .

The two main types of chemistries attributed to ONOO- are oxidations and nitrations. Therefore it is not very surprising that the two main products that have been identified so far from the reaction of dG with ONOO- are 8-oxo-dG and 8-nitro-dG. 8-oxo-dG has long been considered an attractive biomarker for monitoring DNA damage in studies with various oxidizing agents. The role of 8-oxo-dG in mutagenesis and carcinogenesis has been widely investigated and several studies have shown a correlation between the formation of 8-oxo-dG and carcinogenesis. The identification of 8-nitro-dG is especially significant because it provides an analytical tool for the measurement of ONOO- specific DNA damage. Unlike 8-oxo-dG which is known to cause G:C-> A:T transitions no mutagenicity data is available as of yet on 8-nitro-dG. Recent work on the further reactions of 8-oxo-dG with peroxynitrite will be reported. NO-induced mutagenesis. The discovery that phagocytic monocytes and neutrophils can produce NO in addition to reactive oxygen species has led to recent studies of DNA damage and mutations induced by NO and its reactive metabolites under a variety of experimental conditions. Initial investigations involved exposure of target DNA to NO gas, with the following consequences. Exposure of S. typhimurium induced C to T transition mutations, while treatment of nucleosides produced several deamination products (12). Treatment of TK6 human lymphoblastoid cells induced HPRT mutations as well as DNA deamination (xanthine, hypoxanthine) and strand breaks (8). NO gas mutagenized the plasmid pSP189, causing primarily A:T to G:C and G:C to A:T mutations in the supF gene (13).

Treatment of Chinese Hamster Ovary (CHO) cells with NO gas or with peroxynitrite was followed by formation of AP sites and single-strand DNA breaks (9). NO donor drugs have also been used to treat DNA in a variety of experimental models. Treatment of pSP189 with DEA/NO or spermine/NO induced predominantly G:C to A:T mutations (14). Treatment of bronchial epithelial cells with NO donor drugs failed to induce detectable levels of mutation in the HPRT or p53 genes (15). Treatment of pSP189 with peroxynitrite induced strand breaks and supF mutations, predominantly G:C to T:A and G:C to A:T (16).

Study of the DNA damage and mutagenicity of macrophage-derived NO in vivo is essential to assess its potential significance as a genotoxic hazard.

In response to this need, we have developed the SJL-lacZ transgenic animal model, and have produced preliminary evidence of mutagenicity associated with NO overproduction. Similar results have been achieved for mutations in the endogenous HPRT gene in RAW 264.7 macrophages. The potential for DNA damage and mutation by NO and its products is indisputable at the present time.

1. Tannenbaum, S.R., et al. (1994) IN: Nitrosamines and Related N-Nitroso Compounds. Amer. Chem. Soc., Washington, DC, pp. 120-135.

Peroxynitrite is the product of the fast combination reaction between two radical species, superoxide (O2.-) and nitric oxide (.NO). The rate constant for this reaction is close to the diffusion-controlled limit, with a value of about 7 x 109 M-1s-1 (1). While under normal

physiological conditions the catalytic action of cytosolic, mitochondrial and extracellular superoxide dismutases (SOD), rapidly dismutates O2.- to hydrogen peroxide (H2O2) and molecular oxygen (kSOD= 1-2x109 M-1s-1), high levels of .NO can outcompete the dismutation reaction, leading to the formation of peroxynitrite anion (ONOO-). In

fact, upon the discovery of .NO as the endothelial-derived relaxation factor it was established that the presence of SOD increased its biological half-life (2). Thus, while O2.- will limit the physiological functions of .NO, it will also lead to the formation of a secondary species, which has been revealed to have strong oxidizing and cytotoxic potential (3-5).

The formation of ONOO- has provided an alternative molecular mechanism to the Haber-Weiss and Fenton reactions for the biological toxicity ascribed to O2.- (4). Similarly, it has become apparent that.NO-dependent toxicity can not be solely attributed to direct reactions of .NO with biomolecules and tissue oxidative damage exerted by .NO relies in the formation of secondary species. Thus, ONOO- constitutes a common final pathway for oxidative events occurring when .NO and O2.- are cogenerated.

Peroxynitrite anion (ONOO-) is in rapid protonation equilibrium with peroxynitrous acid (ONOOH) (pKa = 6.8) and both species have unique reactivities towards biomolecules. Relevant biological reactions include one- and two-electron oxidations and nitrations. In some cases, these reactions are either catalyzed or mediated through transition metal centers and/or carbon dioxide.

The "peroxynitrite hypothesis" of oxidative injury predicts that cell or tissue protection should be afforded by the use of nitric oxide synthase inhibitors or SOD (or SOD-mimics) and scavengers of ONOO-.

In phosphate buffer, ONOO- decays through a proton-catalyzed isomerization to nitrate (NO3-) with a half-life of 0.6-0.7 s at 37oC and pH 7.4. (k1' = 1 s-1). The rearrangement of ONOOH to NO3- may occur through the transient formation of a caged radical pair [NO2...OH], a fraction of which may escape giving rise to hydroxyl radicals (.OH) and nitrogen dioxide (.NO2). There is no complete agreement in the literature in regard to the intimate mechanism of ONOO- evolution to NO3- (6,7). However, it is clear that during this process oxidants with

reactivity similar to .OH can be formed, as detected experimentally by product formation and competition kinetic analysis with .OH scavengers (3) as well as with EPR-spin trapping-based techniques (8). Oxidation yields by this pathway are usually low, always less than 30 % of initial oxidant added.

Peroxynitrite decomposition also results in low levels of nitration of aromatic and aliphatic residues. For instance, nitration of aromatic residues such as tyrosine, can occur with up to 6-8 % yields (5).

Hydroxyl radical is the strongest oxidizing intermediate formed in biology, with a Eo' = + 2.3 V and its formation during ONOO- decomposition was initially thought of key relevance for the

understanding of the biological reactivity/toxicity of ONOO- (3). However, the ".OH-pathway" of ONOO- has been revealed to be a quantitative minor route in biology. Indeed, direct reactions with either the anionic or protonated forms of ONOO- appear to be significantly more rapid than the slower route, leading to the unimolecular rearrangement of ONOO- (9). We have estimated that under biological conditions < 5 % ONOO- would evolve to the .OH-pathway.

Reaction with thiols. Peroxynitrite reacts with low and high-molecular weight thiols with rate constants in the range of 1-5 x 103 M-1s-1 at 37oC and pH 7.4 (4). While this rate constant is moderate, the high intracellular concentration of thiols makes this a prevalent route intracellularly. Glutathione exists at an intracellular concentration in the order of 1-10 mM and constitutes a key scavenger of ONOO- while reactions of ONOO- with protein thiols can lead to inactivation of biological activity as has been observed for various thiol-containing enzymes (10). Peroxynitrite reaction with low molecular thiols or vicinal thiols in proteins lead to disulfide bond formation, while reaction with isolated protein thiols give rise to the formation of

higher oxidation states of sulfur such as sulfenic, sulfinic or sulfonic acid derivatives. Peroxynitrite oxidation of thiols may occur preferentially through a two-electron oxidation process (11), but one-electron oxidation to thiyl radical has been also detected, which has been attributed to the reaction of the OH-like oxidant with thiol (8,11). Minor product formation (< 1%) of the reaction of ONOO- with thiols include nitrosated or nitrated derivatives (S-nitroso or S-nitrothiols).

Reaction with transition metal containing centers. Peroxynitrite reacts fast with transition metal centers, including those of iron, copper and manganese. For instance, a key reaction involves the interaction with the 4Fe-4S cluster of mitochondrial aconitase (k =1-2 x 105 M-1s-1, Ref. 12), leading to oxidation and disruption of the cluster to the 3Fe-4S form with the concomitant loss in enzyme activity. The interaction with cytosolic aconitase predisposes to total disassembly of the cluster and activation of IRP-1 activity (13). There are also fast reactions of ONOO- with hemeproteins including oxyhemoglobin, peroxidases and cytochrome c2+ (14). The reactions with hemeproteins typically lead to reversible redox changes in the proteins.

Important interactions involve the reactions of ONOO- with copper and manganese SOD (15, 16). Cu-Zn SOD is resistant to peroxynitrite-mediated inactivation, but it can promote nitration reactions efficiently. On the other hand, Mn-SOD is readily inactivated by ONOO-, a process that contributes to mitochondrial oxidative stress.

In addition to the effects that ONOO- can directly have over the biological function and redox state of a transition metal-containing transition metal center, it is important to note that transition metal catalyze nitration reactions by ONOO- (15,17).

The peroxynitrite-carbon dioxide reaction. Bicarbonate is abundant in biological compartments and it is in equilibrium with carbon dioxide (pKa'= 6.1). Carbon dioxide reacts fast with ONOO- (k = 5.8 x 104M-1s-1 at 37oC, Ref. 18) to yield an adduct, the nitrosoperoxocarbonate (ONOOCO2-) (19). Due to the 1-2 mM concentration of CO2 in intra- and extracellular compartments, the formation of ONOOCO2- is a major route of peroxynitrite reactivity in vivo. Nitrosoperoxocarbonate is a reactive intermediate, which redirects ONOO- reactivity towards nitration. Indeed, the presence of CO2 in biological systems significantly increases peroxynitrite-mediated nitrations (18).

In the presence of target biomolecules, the biological half-life of ONOO- could be in the range of 20 ms. This time is long enough to allow for diffusion of ONOO- through biological compartments.

It has been recently shown that ONOO- can cross cell membranes via anion channels while ONOOH could due this through passive diffusion (19). Both mechanisms possibly coexist in biology, with the relative importance of each pathway depending, among other factors, on a combination of tissue pH and the relative abundance of anion channels. Thus, ONOO- could exert biological effects at a moderate distance of its site of formation. For instance, ONOO- formed in the intravascular space by the reaction of activated neutrophil-derived .NO and O2.-. could diffuse inside a red blood cell and react with oxy-hemoglobin before being consumed in plasma. Thus, in addition to kinetic factors, the biological fate and actions of ONOO- will be dictated by diffusional events.

On the other hand, the half-life of ONOOCO2- is so short (< 1 us, Ref. 7), that once this species is formed it will react (or decay) within the same compartment where it was formed.

Peroxynitrite is a reactive intermediate that accounts for a significant fraction of O2.- and .NO-dependent toxicity. The participation of ONOO- in pathological conditions such as inflammation, ischemia-reperfusion, sepsis and neurodegeneration has been proposed (20). It is important to note that despite its high oxidizing potential, ONOO- is not an indiscriminate oxidant in biological systems and a relatively small number of biomolecules account for much of its biological reactivity. It is also relevant to consider that changes in cellular and tissue pH as well as CO2 tensions, may significantly influence ONOO- reactivity, and relative extents of oxidation and nitration. At the cellular level, ONOO- reaction with target molecules has profound effects on tyrosine kinase-dependent signal transduction cascades, mitochondrial metabolism and in triggering cell death via either apoptotic or necrotic pathways. A detailed understanding of the biological reactivity of ONOO- allows for the design of scavenger molecules that decompose and attenuate the toxic effects of ONOO-.

1. Padmaja S, and Huie RE (1993) Reaction of .NO with O2.-. Free Radical Res. Commun. 18,195-199.

2. Rubanyi, G.M. and Vanhoutte, P.M. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. (1986) Am. J. Physiol. 250, H822-H827.

3. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A. and Freeman BA (1990) Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87,1620-1624.

4. Radi, R., Beckman, J.S., Bush, K.M. and Freeman, B.A. (1991). Peroxynitrite oxidation of sulfhydryls. J. Biol. Chem. 266:4244-4250.

5. Pryor, W.A. and Squadrito, G.L. (1995) The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268, L699-L722.

6. Koppenol WH, Moreno JJ, Pryor WA, Ischiropoulos H, Beckman JS (1992) Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem Res Toxicol 5,834-842.

7. Merenyi G and Lind J. (1997) Thermodynamics of peroxynitrite and its CO2 adduct. Chem. Res. Toxicol. 10, 1216-1220.

8. Augusto O., Gatti, R.M. and R. Radi.(1994) Spin-trapping studies of peroxynitrite decomposition and 3-morpholinosydnonimine N-ethylcarbamide auto-oxidation. Arch. Biochem. Biophys. 310, 118-125.

9. Radi, R. (1996) Kinetic analysis of the reactivity of peroxynitrite with biomolecules. Methods in Enzymology. 268, 354-366.

10. Rubbo, H., Denicola, A. and Radi, R. (1994) Peroxynitrite inactivates thiol-containing enzymes of Trypanosoma cruzi energetic metabolism and inhibits cell respiration. Arch. Biochem. Biophys. 308: 96-102.

11.Quijano, C., Alvarez, B, Gatti R, Augusto O and Radi R. (1997). On the pathways of peroxynitrite oxidation of sulfhydryls. Biochem. J. 322, 1 67-173.

12. Castro, L., Rodriguez, M. and R. Radi. (1994) Aconitase is readily inactivated by peroxynitrite but not by its precursor nitric oxide. J. Biol. Chem. 269, 29409-29415.

13. Bouton C., Hirling, H., Drapier JC. (1997) Redox modulation of iron regulatory proteins by peroxynitrite. J. Biol. Chem. 272, 19969-19975.

14. Radi R. (1996) Reactions of nitric oxide with metalloproteins. Chem. Res. Toxicol. 9, 828-835.

15. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J.C., Smith, C.D. and Beckman, J.S. (1992) Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch. Biochem. Biophys. 298,431-437.

16. MacMillan-Crow, L.A., Crow, JP, Kerby JD; Beckman JS and Thompson JA (1996). Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc-Natl-Acad-Sci-U-S-A. 93, 11853-11858.

17. Ferrer-Sueta G., Ruiz-Ramirez L. and Radi R.(1997) Ternary copper complexes and Mn(tbap) catalyze peroxynitrite-dependent nitration of aromatics. Chem. Res. Toxicol. 10, 1338-1344.

18. Denicola A, Freeman BA, Trujillo M and Radi R. (1996) Peroxynitrite reaction with carbon/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations Arch Biochem Biophys 332, 49-58.

19. Lymar S.V. and Hurst J.K. (1995) Rapid reaction between peroxynitrite ion and carbon dioxide: implications for biological activity. J. Am. Chem. Soc. 117, 8867-8868.

20. Denicola, A, Souza J and Radi R. (1998) Peroxynitrite diffusion across erythrocyte membranes. Proc. Natl. Acad. Sci. USA 3566-3571.

21. Beckman J and Koppenol W. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am. J. Physiol. 271, C1424- C1437.

The ability of nitric oxide (NO) to bind to coordination sites of transition metals (especially iron) as well as the reactivity of NO-derived nitrosating species with thiols are indispensable keys to understanding the biological roles of NO. Enzymes often have metal(s) or thiol(s) at crucial catalytic or allosteric sites, and a wide range of them undergo modulation of activity upon NO synthesis. Some are activated (e.g., soluble guanylate cyclase) but the majority are inhibited by NO or its derivatives. Here we describe the inhibition of aconitases by NO and NO-derived species, and focus on how NO synthesis affects the two mutually exclusive functions of the cytosolic aconitase: acting as an enzyme or binding mRNAs.

Aconitases, a class of enzymes very sensitive to environmental redox signals.

Aconitases [citrate (isocitrate)hydro-lyases] are monomeric proteins (Mr 68 000-120 000) containing labile [4Fe-4S] clusters. Remarkably, spectroscopic techniques have revealed that the Fe-S center of mitochondrial aconitase (mt-Acn), the most extensively studied, is not involved in oxidation/reduction but is an active center of the enzyme interconvertible between the [4Fe-4S] and [3Fe-4S] forms. Three of the four iron atoms are linked to 3 cysteines of the protein backbone. The fourth iron atom (Fe_a) is thus exposed and can interact with substrates, solvent or exogenous ligands. Crystallographic studies of porcine heart mt-Acn have revealed the presence of 3 structural domains packed around the [4Fe-4S]²⁺ cluster and a fourth domain on the other side connected to the three others by a linker peptide. Crystal structure determination has also shown that the substrate binds directly to the cluster, through Fe_a.

In the citric acid cycle, mt-Acn catalyzes the isomerization of citrate and isocitrate via the dehydration product cis-aconitate. By doing so, it participates in fueling the electron transport chain. Accordingly, we can expect that its inhibition would slow the electron flow, and in turn decrease cellular energy (ATP) production. Diseases associated with defects of mitochondrial energy yield include lactic acidosis, myopathies, cardiac abnormalities and neurodegenerative diseases. Interestingly, a recent study identified mt-Acn as a specific target of oxidative damage during aging. Indeed, mt-Acn is selectively oxidized during aging of the house fly and loss of mt-Acn activity (mediated by fluoroacetate/fluorocitrate) shortens life-span.

However, another scenario may develop, and it is likely that some cells can bypass mt-Acn blockade. In a series of experiments aimed at pinpointing the sites of injury in guinea pig L10 hepatoma cells after coculture with activated murine macrophages, we observed an early inhibition of mt-Acn which did not result in decreased respiration. To explain this observation we published evidence that exogenous a-ketoglutarate derived from the transamination of glutamate can be used as an alternative substrate of citrate to sustain a truncated cycle sufficient to supply the respiration chain with electrons. Accordingly, inactivation of mt-Acn leads to effects beside limitation of energy e.g., accumulation of citrate, which can serve as a precursor of lipids or may participate in iron transport.

Gardner and Fridovich showed that bacterial Acn and mammalian mt-Acn are very sensitive to oxygen and superoxide in vitro. In vivo experiments revealed significantly decreased levels of mt-Acn in the lungs of rats and baboons exposed to hyperoxia. Moreover, mutant mice lacking Mn-SOD, which normally scavenges superoxide produced as a by-product of oxidative phosphorylation, have significantly reduced levels of mt-Acn.

We have shown that the activities of several mitochondrial Fe-S cluster-containing enzymes were inhibited in activated macrophage-injured tumor cells, and that mt-Acn was the most sensitive. It was subsequently demonstrated that macrophages activated to produce NO exhibited the same pattern of inhibitions which were reversible in the presence of an NO synthase (NOS) inhibitor. NO-dependent inhibition of mt-Acn was then found to occur in many cell types able to express NOS2, including hepatocytes, pancreatic cells and epithelial cells. In vitro experiments with purified mt-Acn confirmed that NO inhibits mt-Acn by targeting its Fe-S, as it yields dinitrosyl-iron complexes detectable by electron paramagnetic resonance spectroscopy. Yet debate continues about whether NO itself or a species derived from NO inhibits mt-Acn. It has been claimed that peroxynitrite rather than NO is the reactive species which reacts with mt-Acn. However, a recent study may have reconciled the divergent interpretations by revealing that porcine mt-Acn is sensitive to NO only when the pH is slightly acidic.

Attention to NO-dependent inhibition of mitochondrial enzymes was recently stimulated by the characterization of a mitochondrial NOS (mt-NOS). Whether or not this newly characterized NOS is responsible for all the NO-dependent mitochondrial dysfunctions remains to be seen. It is antigenically related to NOS2 but is constitutively expressed. The latter result must be reconciled with the well-established observation that mt-Acn inhibition requires prior stimulation of cells by the classical NOS2 inducers (cytokines, LPS, ..).

The cytosolic aconitase dilemma: acting as an enzyme or as a trans-regulator?

Interest in aconitases has been heightened by the finding that its versatile molecular structure allows the cytosolic counterpart of mt-Acn to function in vertebrates as a mRNA-binding protein in regulation of intracellular iron. Indeed, cytosolic aconitase (c-Acn) is identical to iron regulatory protein1 (IRP1, formerly named IRE-BP or IRF), a major regulator of iron homeostasis.

Again, the crucial element in the activity of c-Acn/IRP1 is the [4Fe-4S] cluster, which is linked to cys437, cys503 and cys506 and located in the vicinity of the RNA-binding domain. In iron-replete cells, the [4Fe-4S] cluster-containing protein is the major form active as aconitase whose role in cytosol is ill-defined. Conversely, when intracellular iron is low, a cluster-free form of c-Acn/IRP1 binds to specific sequences termed iron-responsive elements (IREs) which form stem-loop structures at the 5'- or 3'-ends of certain mRNAs. Binding at the 5'-end blocks translation whereas binding at the 3'-end enhances mRNA stability. Five IREs are found in the 3' untranslated region of transferrin receptor mRNA and one IRE is located at the 5'-end of ferritin H- and L-chains, erythroid d-aminolevulinate synthase as well as ... mt-Acn mRNAs. The molecular mechanism by which fluctuation of intracellular iron level promotes [4Fe-4S] cluster assembly/disassembly in vivo is unknown. In addition to these two cardinal forms of c-Acn/IRP1, a [3Fe-4S] cluster-containing form can be purified from cells. This protein, even though it binds citrate, has neither aconitase activity nor IRE-binding capacity because of steric hindrance. It could represent a latent form rapidly convertible into the [4Fe-4S] form.

Escherichia coli AcnA and AcnB as well as plant Acn exhibit a high degree of identity (~50%) with human c-Acn/IRP1. However, there is no evidence thus far for a switch from the enzymatic form of Acn to a regulatory form in bacteria or in plants.

The sensitivity of mt-Acn to reactive oxygen species (ROS) and to NO synthesis led us to consider that c-Acn/IRP1 could be regulated by NO. c-Acn/IRP1 activities were thus assessed in several cell types activated to produce NO, in particular macrophages which are specialized in recycling iron from senescent blood red cells.

As largely documented by Gardner and Fridovich, O₂^- inhibits bacterial and mammalian mt-Acn. Furthermore, H₂O₂ inhibits plant Acn. We thus wondered whether ROS were able to convert c-Acn/IRP1 into IRE-binding protein. We noticed that O₂^-or H₂O₂ reduced aconitase activity without increasing its capacity to bind IREs. Our conclusion was that ROS react with Fe_a and yield a [3Fe-4S]-containing c-Acn/IRP1 unable to accommodate IREs. Others have shown that oxidative stress activates IRE binding but confirmed that the effect of ROS is indirect.

In biological systems, peroxynitrite results from the reaction between NO and O₂^-. It is held that upon exposure to inflammatory stimuli, certain cell types, in particular phagocytes, can produce peroxynitrite. Further, there is an increasingly widespread view that NOS, at least when L-arginine and tetrahydrobiopterin are limiting, can produce peroxynitrite. Lastly, mitochondria can generate O₂^-(as a by-product of respiration) and NO (from mt-NOS). As peroxynitrite is a powerful oxidant able to react with Fe-S clusters and thiols, we anticipated that peroxynitrite could knock out the Fe-S cluster of c-Acn/IRP1 and in turn activate its IRE-binding capacity. However, neither peroxynitrite added as a bolus nor exposure to SIN-1, which can be considered as a peroxynitrite donor in vitro, was able to activate binding to IREs even though each inhibited c-Acn/IRP1 aconitase activity. We then observed that cluster-free recombinant c-Acn/IRP1 loses spontaneous IRE-binding capacity after exposure to peroxynitrite but that the cys437-to-ser437 c-Acn/IRP1 mutant does not. We therefore proposed that peroxynitrite can disrupt the Fe-S cluster of c-Acn/IRP1 without enabling it to accommodate IRE because it oxidizes cys437 and promotes a disulfide bond between this thiol and either cys503 or cys506, two other vicinal thiols in - or next to - the IRE-binding domain of c-Acn/IRP1.

We and others have shown that cytokine and/or lipopolysaccharide-driven NO biosynthesis dramatically increase c-Acn/IRP1 IRE binding in different cell types. As regards stimulation by NO, IRE binding by c-Acn/IRP1 was almost maximal when NOS2 was induced in intact cells. The conversion from the aconitase form to the IRE-binding form could also be elicited in a cell-free system after exposure to various classes of NO-releasing chemicals. However, activation was far from maximal under these conditions. Lastly, we consistently noted that exposure of purified c-Acn/IRP1 resulted in only weak activation. To solve this issue, we tested the capacity of endogenous reducers to cooperate with NO and found that thioredoxin, a protein disulfide reductase, strongly enhanced the RNA-binding activity of NO-treated c-Acn/IRP1. We thus propose that activation of c-Acn/IRP1 proceeds through a two-step reaction: a necessary but insufficient reaction with the cluster and/or the cysteines that hold it to the protein, followed by the reduction of a disulfide bridge by thioredoxin.

With NO and derivatives, the immunological stimuli-dependent NOS2 and the Ca²⁺ -sensitive NOS1 and NOS3 provide cells with useful effectors to achieve reactions at redox-sensitive site(s) of enzymes. mt-Acn and c-Acn/IRP1 are particularly sensitive to redox reactions and to NO synthesis. These two examples lend support to the idea that NO-dependent enzyme inhibition does not necessarily mirror a general "off signal". As regards c-Acn/IRP1, loss of one function benefits another. This is typically regulation rather than toxicity since the reaction is reversible as long as residues of the protein are not oxidized. The interplay between NO and the dynamic Fe-S cluster of aconitases thus represents one of the best examples of the emerging notion of redox signaling.

* As most of the experiments referred to in these comments were performed in the presence of oxygen, it is possible that NO yielded ill-defined reactive species. The term NO will thus be used for simplicity without anticipating the chemical nature of the effector molecule.

Beinert, H., and M. C. Kennedy. 1993. Aconitase, a two-faced protein: enzyme and iron

Bouton, C., H. Hirling, and J. C. Drapier. 1997. Redox modulation of iron regulatory proteins

Castro, L., M. Rodriguez, and R. Radi. 1994. Aconitase is readily inactivated by peroxynitrite,

Drapier, J. C., and J. B. Hibbs, Jr. 1986. Murine cytotoxic activated macrophages inhibit

aconitase in tumor cells. Inhibition involves the iron-sulfur prosthetic group and is reversible.

Drapier, J. C., and J. B. Hibbs, Jr. 1988. Differentiation of murine macrophages to express

nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of

mitochondrial iron-sulfur enzymes in the macrophage effector cells. J Immunol 140:2829-38.

Drapier, J. C., H. Hirling, J. Wietzerbin, P. Kaldy, and L. C. Kuhn. 1993. Biosynthesis of

nitric oxide activates iron regulatory factor in macrophages. Embo J 12:3643-9.

Drapier, J. C., and C. Bouton. 1996. Modulation by nitric oxide of metalloprotein regulatory

Gardner, P. R., and I. Fridovich. 1991. Superoxide sensitivity of the Escherichia coli

Gardner, P. R., G. Costantino, C. Szabo, and A. L. Salzman. 1997. Nitric oxide sensitivity of

Giulivi, C. 1998. Functional implications of nitric oxide produced by mitochondria in

Hausladen, A., and I. Fridovich. 1994. Superoxide and peroxynitrite inactivate aconitases, but

Hentze, M. W., and L. C. Kuhn. 1996. Molecular control of vertebrate iron metabolism:

mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl

Kennedy, M. C., W. E. Antholine, and H. Beinert. 1997. An EPR investigation of the

products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide. J Biol

Klausner, R. D., T. A. Rouault, and J. B. Harford. 1993. Regulating the fate of mRNA: the

Yan, L. J., R. L. Levine, and R. S. Sohal. 1997. Oxidative damage during aging targets

Introduction: Nitric oxide (NO) is a unique biological messenger molecule which mediates diverse physiologic roles. NO is produced from three NO synthase (NOS) isoforms: Neuronal NOS (nNOS), endothelial NOS, and inducible NOS (iNOS). In the central nervous system NO may play important roles in neurotransmitter release, neurotransmitter reuptake, neurodevelopment, synaptic plasticity, cerebral blood flow and regulation of gene expression. However, excessive production of NO following a pathologic insult can lead to neurotoxicity. NO plays a role in mediating neurotoxicity associated with a variety of neurologic disorders, including stroke and Parkinson's Disease.

Nitric Oxide Synthase: NO is formed by the enzymatic conversion of L-arginine by NOS in a catalytic process that consumes five electrons and results in the formation of L-citrulline in the presence of oxygen and NADPH. NOS requires several co-factors including flavin mononucleotide, flavin adenine dinucleotide, heme, and tetrahydrobiopterin. In addition, nNOS and eNOS require calcium and calmodulin, whereas iNOS is calcium independent. Each isoform plays a unique role in pathogenesis of cerebral ischemia.

How is NO neurotoxic? Due to its ability to modulate neurotransmitter release and reuptake, mitochondrial respiration, DNA synthesis, and energy metabolism, it is not surprising that NO is neurotoxic, however, acute toxicity mediated by NO appears to require production of superoxide anion. NO in and of itself is a relatively nontoxic molecule which, in the absence of superoxide anion will not kill cells, even at extremely high concentrations. In the presence of superoxide anion, however, NO is a potent neurotoxin. The reaction of NO with superoxide anion is the fastest biochemical rate constant currently known, resulting in the formation of the potent oxidant, peroxynitrite (ONOO-). It is most likely that ONOO- mediates the toxic activities of NO production. Peroxynitrite is a lipid permeable molecule with a wider range of chemical targets than NO. It can oxidize proteins, lipids, RNA, and DNA.

Neurotoxicity elicited by ONOO- formation may have a dual component. Peroxynitrite is an effective inhibitor of enzymes in the mitochondrial respiratory chain resulting in decreased ATP synthesis and also inhibits the function of manganese SOD (MnSOD) which could lead to increased superoxide anion formation and increased ONOO- formation. Secondly, ONOO- efficiently modifies and breaks DNA strands and inhibits DNA ligase which increases DNA strand breaks. DNA strand breaks activate DNA repair mechanisms including the nuclear enzyme poly(ADP-ribose) polymerase (PARP). PARP catalyzes the attachment of ADP-ribose units from NAD to nuclear proteins such as histone and PARP itself. PARP can add hundreds of ADP-ribose units within seconds to minutes of being activated. For every mole of ADP-ribose transferred from NAD, one mole of NAD is consumed and four free energy equivalents of ATP are required to regenerate NAD to normal cellular levels. Activation of PARP can result in a rapid drop in energy stores which could lead to impaired cellular metabolism and ultimately death.

Stroke: A role for NO in neurotoxicity was first described in an in vitro model of focal ischemia. A five minute exposure to glutamate or NMDA in primary cortical cultures sets in motion a series of events resulting in cell death twelve to eighteen hours later following exposure to these excitatory amino acids. NOS inhibitors, NO scavengers and SOD provide potent neuroprotection in a dose-dependent manner. Furthermore, NO donors elicit neurotoxicity which develops over a similar time course of twelve to eighteen hours. These results have been replicated in in vivo models of focal ischemia using various pharmacologic inhibitors of NOS. There were, however, numerous conflicting reports in in vivo models of focal ischemia which were due to the use of non-selective NOS inhibitors which also affect cerebral blood flow. The development of genetic knockout mice of nNOS or eNOS allowed for elegant studies to be performed to clarify the role of NO derived from nNOS or eNOS in focal ischemia. nNOS knockout mice are dramatically resistant to focal and global ischemia, however, if nNOS knockout mice are treated with nonspecific NOS inhibitors infarct volumes are equivalent to wild-type mice. eNOS knockout mice have greatly increased infarct volumes compared to wild-type mice, however, if eNOS knockout mice are treated with NOS inhibitors infarct volumes are decreased. Therefore, NO derived from nNOS mediates neurotoxicity following focal ischemia, while NO derived from eNOS is critical in maintaining cerebral blood flow and has a positive effect in decreasing infarct volume following focal ischemia.

Activation of PARP is a primary mediator of NMDA or NO neurotoxicity. In vitro pharmacologic inhibition of PARP or genetic knockout of PARP confers dramatic protection against NMDA, NO or oxygen-glucose deprivation mediated toxicity to primary cortical cultures. In vivo, PARP knockout mice are dramatically resistant to focal ischemia. Infarct volumes are decreased over 80% in PARP knockout mice as compared to wild-type mice. Rats treated with the PARP inhibitor, DPQ, have smaller infarct volumes than saline treated rats following focal ischemia. These in vitro and in vivo studies indicate a key role for the activation of PARP in the development of infarction following focal ischemia.

Parkinson's Disease: Parkinson's disease is a movement disorder characterized by the selective loss of dopamine neurons in the substantia nigra which project to the striatum. This human disease can be modeled in mice by injecting the toxin, 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP). MPTP is converted to MPP+ in the nervous system and targets dopaminergic neurons through the high affinity dopamine reuptake transporter. MPP+ is actively transported into mitochondria where it inhibits mitochondrial respiration, in particular complex-I of the electron transport chain resulting in a decrease in ATP production and an increase in superoxide anion formation. It is possible that this increase in superoxide anion production permits the formation ONOO- from endogenous NO. As in stroke and excitotoxicity, production of peroxynitrite can lead to the activation of PARP. Consistent with this hypothesis, nNOS knockout mice are resistant to MPTP-induced neurotoxicity. Levels of the neurotransmitter, dopamine, in the striatum are spared in nNOS knockout mice and neuronal degeneration visualized by silver staining is reduced in nNOS knockout mice treated with selective nNOS inhibitors compared to saline treated controls. Additionally, PARP knockout mice are dramatically resistant to MPTP neurotoxicity as compared to wild-type mice. Dopamine levels in the striatum are spared as are dopamine containing neurons in the substantia nigra in PARP knockout mice.

Cell Survival: NO may play a key role in nervous system morphogenesis and developmental synaptic plasticity. Cerebral subcortical plate neurons transiently express nNOS from embryonic day E15-E19 of rats. These cells extend their processes through to the corpus striatum and thalamus. nNOS positive immunostaining of subcortical plate neurons and their processes decreases rapidly and vanishes by the 15th postnatal day. Development of proper patterns of connections in the retinotectal system may involve NO as nNOS expression peaks at the time when refinement of the initial pattern of connections is occurring.

The molecular mechanism for NO's role in neural development is not known. A key to how NO regulates neuronal growth, differentiation, survival and death may come from recent observations that NO activates p21^Ras (Ras). Our recent studies indicate that stimulation of NMDA receptor in cultured cortical neurons activates Ras-ERK pathway via calcium-dependent activation of nNOS and NO generation through non-cGMP dependent mechanisms. Activation of Ras/ERK pathway by NO may be mediated by direct activation of Ras GTPase activity presumably by nitrosylation or nitration of cysteine through a redox-sensitive interaction. NO may be the key mediator linking activity to gene expression and long-term plasticity as calcium-dependent activation of Ras-ERK pathway is thought to be a major pathway of neural activity-dependent long-term changes in the nervous system. Thus, NO-Ras signaling may underlie NO's role in neuronal survival, differentiation and apoptotic cell death during development. These processes may occur through redox-sensitive modulation of Ras and suggest that Ras is a potential endogenous NO-redox sensitive effector molecule mediating the intercellular actions of NO in the CNS.

Conclusion: NO has revolutionized our concepts about neuronal signaling. Recent advances indicate that NO's actions are more diverse then originally appreciated. Uncovering the targets of NO and the mechanisms that regulate the expression of nNOS will contribute to a greater understanding of CNS physiology and pathological neuronal functions.

There is increasing evidence that endogenous nitric oxide (NO) plays a key role in physiological regulation of airway functions and is implicated in inflammatory airway diseases, including asthma [1-3].

Immunohistological studies have identified the presence of all three isoforms of NOS in human airways. eNOS is localised to endothelial cells in the bronchial circulation, but there is also evidence for eNOS expression in epithelial cells [4]. nNOS is localised to nerves in airways which are cholinergic [5], but has also been reported in epithelial cells [6]. iNOS may be expressed in several types of cell in response to cytokines, endotoxin or oxidants [7]. In asthmatic airways, there is increased immunocytochemical staining for iNOS, which is localised predominantly to airway epithelial cells [8] and there is also localisation to inflammatory cells in asthmatic airways, including macrophages and eosinophils [9].

NO may be produced by several cells in the airways. In primary cultured human airway epithelial cells pro-inflammatory cytokines increase NO production and increase iNOS immunoreactivity and mRNA [6,10,11]. In a human epithelial cell line (A549) and in rat type II pneumocytes oxidants and ozone increase iNOS expression [12,13]. This is associated with activation of NF-B, which is involved in the transcription of many inflammatory and immune genes [14]. NF-B is of critical importance in increasing the transcription of the iNOS gene [15] and may be activated in several types of pulmonary cell by pro-inflammatory cytokines. Glucocorticoids inhibit the induction of iNOS in epithelial cells and this is likely to be via a direct inhibitory interaction with NF-B. Eosinophils also express iNOS and release nitrite [16]. It has proved difficult to induce iNOS in human, compared to rodent, macrophages. In human monocytes anti-CD23 antibody causes release of nitrite, suggesting that allergens may trigger iNOS expression [17] and similar results are seen in alveolar macrophages form normal and asthmatic subjects [18].

NO is rapidly transformed to nitrite and nitrate, which may be used to monitor NO production. NO also rapidly combines with superoxide anions (O₂^-) to form peroxynitrite (ONOO^-), which is highly reactive and nitrosylates proteins. The presence of nitrotyrosine has recently been demonstrated in asthmatic airways, providing evidence for peroxynitrite generation within the airways and the amount of nitrotyrosine immunostaining is correlated with airway hyperresponsiveness, as measured by methacholine challenge [9].

NO has many effects on airway function, although the effects of endogenous NO depend on the site of production and on the amount produced [19].

NO and NO donor compounds relax human airway smooth muscle in vitro via activation of guanylyl cyclase and an increase in cyclic GMP [20,21]. High concentrations of inhaled NO produce bronchodilatation and protect against cholinergic bronchoconstriction in guinea pigs in vivo [22]. In humans inhalation of high concentrations of NO (80 ppm) has no effect on lung function in normal subjects and produces only weak and variable bronchodilatation in asthmatic patients [23-25]. NO may however be the major neurotransmitter of bronchodilator nerves in human airways. In proximal human airways there is a prominent inhibitory non-adrenergic non-cholinergic (i-NANC) bronchodilator neural mechanism, which assumes particular functional importance as it is the only endogenous bronchodilator pathway in human airways. The neurotransmitter of this i-NANC pathway in human airways is NO, since NOS inhibitors virtually abolish this neural response [26-28]. Furthermore, i-NANC stimulation of human airways results in an increase in cyclic GMP without any increase in cyclic AMP [20]. The density of nNOS-immunoreactive nerves is greatest in proximal airways and diminishes peripherally, which is consistent with a reduction in i-NANC responses in more peripheral airways [29]. NOS is predominantly localised to parasympathetic (cholinergic) nerves and may be co-localised with vasoactive intestinal polypeptide (VIP), although the functional role of endogenous VIP in human airways is obscure [27].

NO is a potent vasodilator in the bronchial circulation and may play an important role in regulating airway blood flow, as in the pulmonary circulation [30-33]. Endogenous NO may increase the exudation of plasma by increasing blood flow to leaky post-capillary venules, thus increasing airway edema [34]. However, NOS inhibitors applied to the airway surface increase plasma exudation, suggesting that basal release of NO has an inhibitory effect on microvascular leakage [35]. This paradox is resolved by the differing effects of NO depending on the amount produced. Thus in rat airways L-NAME increases basal leakiness whereas after endotoxin exposure, when iNOS is induced, L-NAME inhibits leakage [36]. Thus, the effect of endogenous NO on plasma exudation may depend on the amount produced and the site of production. In the context of asthma the increased production of NO is likely to result in increased plasma exudation. Furthermore, if peroxynitrite is generated in asthma this may lead to the formation of hydroxyl radicals that also increase airway plasma exudation [37].

L-NAME increases baseline airway mucus secretions, suggesting that NO derived from cNOS normally inhibits mucus secretion [38]. However NO donors increase mucus secretion in human airways in vitro [39]. In cultured guinea-pig airways after exposure to TNF- and other inflammatory stimuli there is increased secretion of mucus, which is inhibited by L-NMMA, suggesting that large amounts of NO generated by iNOS stimulate mucus secretion [40]. Endogenous NO may also be important in regulating mucociliary clearance, since an NOS inhibitor decreases ciliary beat frequency in bovine airway epithelial cells [41].

NO is the neurotransmitter of bronchodilator nerves in human airways, as discussed above. NO may be co-released with acetylcholine from cholinergic nerves and may modulate cholinergic neural responses. NOS inhibitors increase cholinergic neural bronchoconstriction in human and guinea-pig airways [42-44]. However, this appears to be due to functional antagonism at the level of airway smooth muscle, rather than an effect on ACh release from cholinergic nerves [44,45].

There is increasing evidence that high concentrations of NO may have effects on the immune system and the inflammatory response. NO inhibits Th1 lymphocytes in mice and thus favors the development of a Th2 response with eosinophilia [46,47]. There is also evidence that NO promotes the chemotaxis of eosinophils, since L-NAME blocks eosinophil recruitment in the lungs [48]. NO-donor compounds increase the survival of eosinophils by inhibiting apotosis [49] and NO inhibits Fas-receptor mediated apoptosis in these cells [50]..

There is evidence for increased expression of iNOS in asthmatic airways, particularly in epithelial cells and macrophages [8,9]. It is likely that this arises from the effects of proinflammatory cytokines, oxidants and perhaps other inflammatory mediators. Since NO is a gas it diffuses into the airway lumen and may be detected in exhaled air [51]. There is an increase in exhaled NO in the exhaled air of asthmatic patients [52-54], which is derived from the lower airways [55,56]. The increased exhaled NO in asthma is related to airway inflammation [57], is increased during the late response to allergen [58] and during exacerbations [59], and is decreased by treatment with inhaled corticosteroids [60].

NO is detectable in the exhaled air of normal humans and is presumed to arise from cells lining the airways. NO may be detected in exhaled air by chemiluminescence analysers. Much of the NO in exhaled air of normal individuals may arise from the upper respiratory tract. The levels of NO are increased in the exhaled air of patients with asthma who are not treated with steroids. The raised levels appear to arise from the lower airways, since similar values are found on direct sampling by a fibreoptic bronchoscope and when nasal contamination of exhaled air is prevented [55,61]. The increased exhaled NO in asthma is likely to be derived from iNOS expression in the airways, which has been localised to airway epithelial cells and macrophages. There is a correlation between exhaled NO and iNOS expression in epithelial and inflammatory cells of the airways in asthmatic patients, and also with the amount of nitrotyrosine immunoreactivity, which reflects peroxynitrite formation. Furthermore, exhaled NO is correlated with the proportion of eosinophils in induced sputum and in biopsies. Exhaled NO is further increased in association during the late (inflammatory) response to allergen in asthmatic patients, suggesting that it may reflect airway inflammation [58] Furthermore, exhaled NO is increased during exacerbations of asthma and when inhaled steroid doses are reduced [62]. Exhaled NO levels are normal in asthmatic patients treated with inhaled steroids, providing further evidence that the increased exhaled NO is derived from iNOS, since steroids inhibit iNOS but not constitutive NOS [53]. Controlled studies have demonstrated that exhaled NO is reduced in asthmatic patients by oral and inhaled steroids and by inhaled NOS inhibitors (L-NMMA, L-NAME, aminoguanidine), whereas inhaled ₂-agonist bronchodilators have no such effect [60,63,64].

NO is a potent vasodilator and may increase plasma exudation. It may also participate in the inflammatory response by tipping the balance towards Th2 cells, and by recruiting and increasing the survival of eosinophils in the airways [47].

While exhaled NO is a useful non-invasive marker of inflammation in asthma, it is less certain how endogenous NO contributes to the pathophysiology of asthma. Single inhalations of L-NMMA and L-NAME (via a nebuliser) result in reduced exhaled NO in normal and asthmatic patients [53,63,65]. Interestingly, there is no fall in FEV₁, even in asthmatic patients with highly reactive airways, suggesting that basal production of NO is not important in basal airway tone. Although infusion of L-NMMA in normal subjects causes an increase in blood pressure, neither nebulised L-NAME nor L-NMMA have any effect on heart rate or blood pressure, suggesting that inhibition of NOS is confined to the respiratory tract. While L-NMMA and L-NAME are non-selective inhibitors of constitutive NOS and iNOS, aminoguanidine has some selectivity for iNOS. Inhalation of aminoguanidine has no effect on exhaled NO in normal subjects, but significantly reduces exhaled NO in patients with asthma [63], adding further support to the view that the elevated exhaled NO in asthma is derived from iNOS. More potent and selective iNOS inhibitors are now in clinical development [66].

Exhaled NO is elevated in virally-induced upper respiratory tract infections, which may reflect the expression of iNOS in response to rhinovirus-induced NF-B activation [67]. Exhaled NO is also increased in bronchiectasis and is related to the extent of lung involvement [68]. Surprisingly exhaled NO is reduced in cystic fibrosis [69] and this may be due to the intense neutrophilic inflammation, so that NO is consumed by superoxide anions into peroxynitrite. In chronic obstructive lung disease exhaled NO is much lower than in asthma despite the fact that there is an active inflammatory process [70]. This may be due to the effects of cigarette smoking, which lowers exhaled NO concentrations [71], and due to the neutrophilic inflammation. Exhaled NO may also be elevated in lung parenchymal inflammation, and there is an increase in exhaled NO in patients with active fibrosing alveolitis, but when fibrosis intervenes exhaled NO levels may fall [72], suggesting that exhaled NO may be used as a marker of disease activity. This is consistent with histological studies demonstrating increased iNOS expression during the active inflammatory phase of the disease, with no iNOS expression in areas of fibrosis [73].

1. Barnes PJ, Belvisi MG. Nitric oxide and lung disease. Thorax 1993; 48: 1034-1043.

2. Gaston B, Drazen JM, Loscalzo J, Stamler JS. The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 1994; 149: 538-551.

4. Shaul PW, North AJ, Wu LC, et al. Endothelial nitric oxide synthase is expressed in cultured bronchiolar epithelium. J Clin Invest 1994; 94: 2231-2236.

5. Fischer A, Mundel P, Mayer B, Preissler U, Philippin B, Kummer W. Nitric oxide synthase in guinea-pig lower airway innervation. Neurosci Lett 1993; 149: 157-160.

6. Asano K, Chee CBE, Gaston B, Lilly CM, Gerard C, Drazen JM, Stamler JS. Constitutive and inducible nitric oxide synthase gene expression, regulation and activity in human lung epithelial cells. Proc Natl Acad Sci USA 1994; 91: 10089-10093.

7. Morris S, Billiar TR. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol 1994; 266: E829-839.

8. Hamid Q, Springall DR, Riveros-Moreno V, et al. Induction of nitric oxide synthase in asthma. Lancet 1993; 342: 1510-1513.

9. Giaid A, Saleh D, Lim S, Barnes PJ, Ernst P. Formation of peroxynitrite in asthmatic airways. FASEB J 1998 (in press).

10. Robbins RA, Barnes PJ, Springall DR, et al. Expression of inducible nitric oxide synthase in human bronchial epithelial cells. Biochem Biophys Res Commun 1994; 203: 209-218.

11. Guo FH, de Raeve HR, Rice TW, Stuehr DJ, Thunnissen FBJM, Erzurum SC. Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. Proc Natl Acad Sci USA 1995; 92: 7809-7813.

12. Adcock IM, Brown CR, Kwon OJ, Barnes PJ. Oxidative stress induces NF-kB DNA binding and inducible NOS mRNA in human epithelial cells. Biochem Biophys Res Commun 1994; 199: 1518-1524.

13. Punjabi CJ, Laskin JD, Pendino KJ, Goller NL, Durham SK, Laskin DL. Production of nitric oxide by rat type II pneumocytes: increased expression of inducible nitric oxide synthase following inhalation of a pulmonary irritant. Am J Resp Cell Mol Biol 1994; 11: 165-172.

14. Barnes PJ, Karin M. Nuclear factor-B: a pivotal transcription factor in chronic inflammatory diseases. New Engl J Med 1997; 336: 1066-1071.

15. Xie Q, Kashiwarbara Y, Nathan C. Role of transcription factor NF-kB/Rel in induction of nitric oxide synthase. J Biol Chem 1994; 269: 4705-4708.

16. del Pozo V, de Arruda Chaves E, de Andres B, et al. Eosinophils transcribe and translate messenger RNA for inducible nitric oxide synthase. J Immunol 1997; 158: 859-864.

17. Aubry JP, Dugas N, Lecoanet Henchoz S, et al. The 25-kDa soluble CD23 activates type III constitutive nitric oxide-synthase activity via CD11b and CD11c expressed by human monocytes. J Immunol 1997; 159: 614-622.

18. Donnelly LE, Hart LA, Lim S, Chung KF, Barnes PJ. Increased production of nitrite from human alveolar macrophages following stimulation with CD23. Am J Respir Crit Care Med 1998; 157: A712

20. Ward JK, Barnes PJ, Tadjkarimi S, Yacoub MH, Belvisi MG. Evidence for involvement of cGMP in neural bronchodilator responses in human trachea. J Physiol 1995; 483: 525-536.

21. Gaston B, Reilly J, Drazen JM, et al. Endogenous nitrogen oxides and bronchodilator S-nitrosolthiols in human airways. Proc Natl Acad Sci USA 1993; 90: 10957-10961.

22. Dupuy PM, Shore SA, Drazen JM, Frostell C, Hill WA, Zapol WM. Bronchodilator action of inhaled nitric oxide in guinea pigs. J Clin Invest 1992; 90: 421-428.

23. Högman M, Frostell CG, Hedenström H, Hedenstierna G. Inhalation of nitric oxide modulates adult human bronchial tone. Am Rev Respir Dis 1993; 148: 1474-1478.

24. Sanna A, Kurtansky A, Veriter C, Stanescu D. Bronchodilator effect of inhaled nitric oxide in healthy men. Am J Respir Crit Care Med 1994; 150: 1702-1709.

25. Kacmarek RM, Ripple R, Cockrill BA, Bloch KJ, Zapol WM, Johnson DC. Inhaled nitric oxide: a bronchodilator in mild asthmatics with methacholine-induced bronchospasm. Am J Respir Crit Care Med 1996; 153: 128-135.

26. Belvisi MG, Stretton CD, Barnes PJ. Nitric oxide is the endogenous neurotransmitter of bronchodilator nerves in human airways. Eur J Pharmacol 1992; 210: 221-222.

27. Belvisi MG, Stretton CD, Miura M, Verleden GM, Tadjarimi S, Yacoub MH, Barnes PJ. Inhibitory NANC nerves in human tracheal smooth muscle: a quest for the neurotransmitter. J Appl Physiol 1992; 73: 2505-2510.

28. Bai TR, Bramley AM. Effect of an inhibitor of nitric oxide synthase on neural relaxation of human bronchi. Am J Physiol 1993; 264: L425-L430.

29. Ward JK, Belvisi MG, Springall DR, et al. Human iNANC bronchodilatation and nitric oxide-immunoreactive nerves are reduced in distal airways. Am J Resp Cell Mol Biol 1995; 13: 175-184.

30. Higenbottam TW. Lung disease and pulmonary endothelial nitric oxide. Exp Physiol 1995; 134: 855-864.

31. Crawley DF, Liu SF, Evans TW, Barnes PJ. Inhibitory role of endothelium-derived nitric oxide in rat and human pulmonary arteries. Br J Pharmacol 1990; 101: 166-170.

32. Liu SF, Crawley DE, Barnes PJ, Evans TW. Endothelium derived nitric oxide inhibits pulmonary vasoconstriction in isolated blood perfused rat lungs. Am Rev Respir Dis 1991; 143: 32-37.

33. Martinez C, Cases E, Vila JM, Aldasoro M, Medina P, Marco V, Lluch S. Influence of endothelial nitric oxide on neurogenic contraction of human pumlonary arteries. Eur Respir J 1995; 8: 1328-1332.

34. Kuo H, Liu S, Barnes PJ. The effect of endogenous nitric oxide on neurogenic plasma exudation in guinea pig airways. Eur J Pharmacol 1992; 221: 385-388.

35. Erjefält JS, Erjefält I, Sundler F, Persson CGA. Mucosal nitric oxide may tonically suppress airway plasma exudation. Am J Resp Crit Care Med 1994; 150: 227-232.

36. Bernareggi M, Mitchell JA, Barnes PJ, Belvisi MG. Dual action of nitric oxide on airway plasma leakage. Am J Respir Crit Care Med 1997; 155: 869-874.

37. Lei Y-H, Barnes PJ, Rogers DF. Involvement of hydroxyl radicals in neurogenic airway plasma exudation and bronchoconstriction in guinea pigs in vivo. Br J Pharmacol 1996; 117: 449-454.

38. Ramnarine SI, Khawaja AM, Barnes PJ, Rogers DF. Nitric oxide inhibition of basal and neurogenic mucus secretion. Br J Pharmacol 1996; 118: 998-1002.

39. Nagaki M, Shimura MN, Irokawa T, Sasaki T, Shirato K. Nitric oxide regulation of glycoconjugate secretion from feline and human airways in vitro. Respir Physiol 1995; 102: 89-95.

40. Adler KB, Fischer BN, Li H, Choe NH, Wright DT. Hypersecretion of mucin in response to inflammatory mediators by guinea pig tracheal epithelial cells in vitro is blocked by inhibition of nitric oxide synthase. Am J Respir Cell Mol Biol 1995; 13: 526-530.

41. Jain B, Lubinstein I, Robbins RA, Leise KL, Sisson JH. Modulation of airway epithelial cell ciliary beat frequency by nitric oxide. Biochem Biophys Res Commun 1993; 191: 83-88.

42. Belvisi MG, Stretton CD, Barnes PJ. Nitric oxide as an endogenous modulator of cholinergic neurotransmission in guinea pig airways. Eur J Pharmacol 1991; 198: 219-221.

43. Belvisi MG, Miura M, Stretton CD, Barnes PJ. Endogenous vasoactive intestinal peptide and nitric oxide modulate cholinergic neurotransmission in guinea pig trachea. Eur J Pharmacol 1993; 231: 97-102.

44. Ward JK, Belvisi MG, Fox AJ, Miura M, Tadjkarimi S, Yacoub MH, Barnes PJ. Modulation of cholinergic neural bronchoconstriction by endogenous nitric oxide and vasoactive intestinal peptide in human airways in vitro. J Clin Invest 1993; 92: 736-743.

45. Brave SR, Hobbs AJ, Gibson A, Tucker JF. The influence of L-N^G-nitro-arginine on field stimulation induced contractions and acetylcholine release in guinea pig isolated tracheal smooth muscle. Biochem Biophys Res Commun 1991; 179: 1017-1022.

46. Taylor-Robinson AW, Phillips RS, Severin A, Moncada S, Liew FY. The role of TH1 and TH2 cells in a rodent malaria infection. Science 1993; 260: 1931-1934.

47. Barnes PJ, Liew FY. Nitric oxide and asthmatic inflammation. Immunol Today 1995; 16: 128-130.

48. Ferreira HHA, Medeiros MV, Lima CSP, Flores CA, Sannomiya P, Antunes E, De Nucci G. Inhibition of eosinophil chemotaxis by chronic blockade of nitric oxide biosynthesis. Eur J Pharmacol 1996; 310: 201-207.

49. Beauvais F, Michel L, Dubertret L. The nitric oxide donors, azide and hydroxylamine, inhibit the programmed cell death of cytokine-deprived eosinophils. FEBS Lett 1995; 361: 229-232.

50. Hebestreit H, Dibbert B, Balatti I, Braun D, Schapowal A, Blaser K, Simon H-U. Disruption of Fas receptor signalling by nitric oxide in eosinophils. J Exp Med 1998; 187: 415-425.

51. Barnes PJ, Kharitonov SA. Exhaled nitric oxide: a new lung function test. Thorax 1996; 51: 218-220.

52. Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993; 6: 1268-1270.

53. Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne E, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994; 343: 133-135.

54. Persson MG, Zetterstrom O, Argenius V, Ihre E, Gustafsson LE. Single-breath oxide measurements in asthmatic patients and smokers. Lancet 1994; 343: 146-147.

55. Kharitonov S, Chung KF, Evans DJ, O'Connor BJ, Barnes PJ. Increased exhaled nitric oxide in asthma is derived from the lower respiratory tract. Am J Respir Crit Care Med 1996; 153: 1773-1780.

56. Massaro AF, Mehta S, Lilly CM, Kobzik L, Reilly JJ, Drazen JM. Elevated nitric oxide concentrations in isolated lower airway gas of asthmatic subjects. Am J Respir Crit Care Med 1996; 153: 1510-1514.

57. Jatakanon A, Lim S, Kharitonov SA, Chung KF, Barnes PJ. Correlation between exhaled nitric oxide, sputum eosinophils and methacholine responsiveness. Thorax 1998; 53: 91-95.

58. Kharitonov SA, O'Connor BJ, Evans DJ, Barnes PJ. Allergen-induced late asthmatic reactions are associated with elevation of exhaled nitric oxide. Am J Resp Crit Care Med 1995; 151: 1894-1899.

59. Massaro AF, Gaston B, Kita D, Fanta C, Stamler J, Drazen JM. Expired nitric oxide levels during treatment for acute asthma. Am J Respir Crit Care Med 1995; 152: 800-803.

60. Kharitonov SA, Yates DH, Barnes PJ. Regular inhaled budesonide decreases nitric oxide concentration in the exhaled air of asthmatic patients. Am J Resp Crit Care Med 1996; 153: 454-457.

61. Kharitonov SA, Barnes PJ. Nasal contribution to exhaled nitric oxide during exhalation against resistance or during breath holding. Thorax 1997; 52: 540-544.

62. Kharitonov SA, Yates DH, Chung KF, Barnes PJ. Changes in the dose of inhaled steroid affect exhaled nitric oxide levels in asthmatic patients. Eur J Respir Dis 1996; 9: 196-201.

63. Yates DH, Kharitonov SA, Worsdell M, Thomas PS, Barnes PJ. Exhaled nitric oxide is decreased after inhalation of a specific inhibitor of inducible nitric oxide synthase, in asthmatic but not in normal subjects. Am J Resp Crit Care Med 1996; 154: 247-250.

64. Yates DH, Kharitonov SA, Barnes PJ. Effect of short- and long-acting b₂-agonists on exhaled nitric oxide in asthmatic patients. Eur Respir J 1997; 10: 1483-1488.

65. Yates DH, Kharitonov SA, Robbins RA, Thomas PS, Barnes PJ. Effect of a nitric oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide. Am J Resp Crit Care Med 1995; 152: 892-896.

66. Garvey EP, Oplinger JA, Furfine ES, Kiff RJ, Laszlo F, Whittle BJR, Knowles RG. 1400W is a slow tight binding and highly selective inhibitor of inducble nitric oxide synthase in vitro and in vivo. J Biol Chem 1997; 272: 4959-4963.

67. Kharitonov SA, Yates D, Barnes PJ. Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections. Eur Resp J 1995; 8: 295-297.

68. Kharitonov SA, Wells AU, O'Connor BJ, Hansell DM, Cole PJ, Barnes PJ. Elevated levels of exhaled nitric oxide in bronchiectasis. Am J Resp Crit Care Med 1995; 151: 1889-1893.

69. Balfour-Lynn IM, Laverty A, Dinwiddie R. Reduced upper airway nitric oxide in cystic fibrosis. Arch Dis Child 1996; 75: 319-322.

70. Maziak W, Loukides S, Culpitt S, Sullivan P, Kharitonov SA, Barnes PJ. Exhaled nitric oxide in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 157: 998-1002.

71. Kharitonov SA, Robbins RA, Yates D, Keatings V, Barnes PJ. Acute and chronic effects of cigarette smoking on exhaled nitric oxide. Am J Resp Crit Care Med 1995; 152: 609-612.

72. Paredi P, Kharitonov SA, Maziak W, du Bois RM, Barnes PJ. Exhaled nitric oxide (NO) a possible marker for disease activity in patients with interstitial lung disease. Eur Respir J 1997; 10(suppl 25): 158S

73. Saleh D, Barnes PJ, Giaid A. Increased production of the potent oxidant peroxynitire in the lungs of patients with pulmonary fibrosis. Am J Respir Crit Care Med 1997; 155: 1763-1769.

Experimental evidence of selective pulmonary vasodilation with inhaled nitric oxide (iNO) in sheep was published in 1991 (1). An earlier preliminary report on a similar effect in humans with established pulmonary hypertension was followed up the same year with a full paper (2). Subsequently, iNO has been a clinical fact since the first exposures of patients with severe hypoxemia and pulmonary hypertension in 1991-92 (3,4,5). Early on it became clear that iNO results in reduction of pulmonary hypertension caused by vasospasm, and improvement in oxygen exchange in a majority of patients with acute lung injury (ALI). The latter effect is more unpredictable, as only about 60% of patients with ALI display an improvement over the first few hours (6,7). Experimental and clinical research suggest that alveolar recruitment, pulmonary perfusion and presence of pulmonary vasoconstriction influences the degree of immediate response in terms of better oxygenation (8). It is at present unclear if a positive response is stable over an extended period of time (several days). An additional complication in clinical studies, is a controversy whether or not acute manipulation of physiological parameters, such as oxygenation, can alter the outcome of the syndrome life-threatening ALI.

Although a gas for inhalation, iNO cannot be accepted as a drug in clinical medicine if not registered as such, proving efficacy and safety. This has prompted an effort to demonstrate efficacy and safety in multicenter studies on a proper indication for iNO. To this point, interest in using iNO as therapy has been focused on the three following clinical situations: (1) in newborns with severe hypoxic respiratory failure and pulmonary hypertension, (2) in acute lung injury (ALI) in older children and adults, and (3) as an additional tool to reduce severe pulmonary hypertension after cardiac surgery in children and adults.

(a) Newborns. Three major studies have given evidence, that iNO acutely improves

oxygenation when administered in severe hypoxic respiratory failure (9) or in PPHN

(Persistent pulmonary hypertension of the newborn, 10,11). Also the need for ECMO was

(b) ALI. Recently three major randomised studies have presented data on clinical use of iNO with the aim to improve outcome. An American phase II, blinded and placebo-controlled study conducted by Ohmeda included 177 patients, comparing 1.25 -5 -10 -20 -40 and 80 parts per million (ppm) of NO with control (6). More than 60% of patients were `responders' in so far that arterial oxygenation improved >20%, however also some placebo patients were responders at the 4 hour post randomisation point. Mortality and adverse reactions were similar in all groups. Patients alive and extubated by day 28 after randomisation tended to increase (post-hoc analysis) in the subgroup administered 5 ppm NO. An ongoing phase III study is presently randomising control treatment (no iNO) vs 5 ppm NO in patients with isolated severe lung injury during mechanical ventilation. Preliminary results from a European multicenter study is now available as an abstract. The study randomised 180 patients to iNO 2-40 ppm or control (12) before being stopped prematurely due to poor recruitment. The study was not blinded. Preliminary data does not demonstrate increased frequency of reversal of ALI or faster such reversal in the iNO group. However, less patients in the iNO group developed severe respiratory failure, defined as inclusion criteria for ECMO. Significantly more patients in the iNO arm of the study were given renal replacement therapy or displayed increased creatinine (combined endpoint) for unclear reasons. A French multicenter randomised and placebo-controlled study included about 200 patients, and also found minor differences between the NO and placebo treated patients (D Payen, personal communication).

(c) Pulmonary hypertension, in connection with cardiac surgery and more. Anecdotes and small studies suggest that iNO can be used to control severe pulmonary hypertensive reactions in the postsurgical period. A randomised double-blinded recently published study in children with congenital heart disease supplies evidence that iNO in the immediate postoperative phase is a selective pulmonary vasodilator in the subgroup of patients who emerge from cardiopulmonary bypass with pulmonary hypertension (13). In chronic pulmonary hypertension partial reduction of righ ventricular dysfunction can be achieved with iNO (14). In a NO synthase deficient mouse model it was clear that iNO protected from the development of chronic pulmonary hypertension seen in control animals (15 ).

Adverse reactions, safety issues. The dangers and difficulties with inadequate monitoring and delivery devices for administering iNO has been pointed out (17, 18). Uncontrolled exposure to the contaminant NO₂ or error in dosing are obvious risks. Consensus statements have been formulated locally or published as guidelines (19). Intracranial hemorrhage or excessive bleeding was not a major factor in randomised studies cited above. Sudden withdrawal of NO may cause deleterious effects, and careful weaning seems called for. Increased number of chromosome aberrations in blood lymphocytes were not found in volunteers inhaling 40 ppm NO (20). There are conflicting data on the effect of NO on tissue inflammation; protective or not? Humans produce and autoinhale high doses of endogenously produced NO in the nose from birth and onwards (21).

Future aspects. Studies in which iNO is combined with other therapeutic strategies for the treatment of ALI, such as mechanical ventilation in the prone position, permissive hypercapnia (low tidal volume leading to relative hypoventilation), high-frequency oscillation, partial liquid ventilation, administration of surfactant, or a facilitator of hypoxic vasoconstriction (almitrine); are all being performed. On the other hand some researchers instead advocate aerosolized prostaglandins as selective vasodilators due to unsolved toxicity issues with iNO. Recently presented smaller clinical studies confirm that similar results on physiological parameters can be obtained as with iNO (22). There are additional ways to manipulate the L-arginine/NO pathway than just administering iNO. Ichinose et al studied lambs inhaling a phosphodiesterase inhibitor Zaprinast with and without iNO and found synergistic effects (23). Apart from manipulations of pulmonary vascular resistance and gas exchange, researchers are exploring other effects of gaseous NO. By adding NO to the sweep gas of membrane oxygenators during extracorporeal circulation, an anticoagulative effect of NO is exploited, which in turn reduces platelet consumption during such bypass procedures (24).

Summary Randomised clinical studies in hypoxic newborns demonstrate that oxygenation is improved and the need for ECMO reduced by the addition of iNO. Long term follow-up studies on treated patients will hopefully soon be available. In contrast, randomised studies in adult severe acute lung injury do not support the general use of iNO in that clinical situation. Randomised studies on other conditions are scarce. Smaller pilot studies are exploring a multitude of combination therapy approaches, into which the pharmacodynamic effect of causing selective pulmonary vasodilation with iNO is integrated.

Acknowledgements This work was in part made possible by a grant from the Swedish Medical Research Council (no 9073). The author wishes to disclose that he has participated in patent applications for the clinical use of inhaled NO, and that he works part time as consultant to industry related to NO gas production.

1. Frostell CG, Fratacci M-D, Wain JC, Zapol WM: Inhaled nitric oxide: a selective pulmonary

vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation 1991;83: 2038-2047

2. Pepke-Zaba J, Higenbottam TW, Dinh-Xuan AT, Stone D, Wallwork J: Inhaled nitric oxide

4. Kinsella JP, Neish SR, Shaffer E, Abman SH: Low-dose inhalational nitric oxide in

5. Roberts JD, Polaner DM, Lang P, Zapol WM: Inhaled nitric oxide in persistent pulmonary

6. Dellinger RP, Zimmerman JL, Taylor RW, et al: Effects of inhaled nitric oxide in

patients with acute respiratory distress syndrome: Results of a randomized phase II trial.

7. Lundin S, Nathorst Westfelt U, Stenqvist O, Blomqvist H, Lindh A, Berggren L, Arvidsson

S, Rudberg U, Frostell CG: Response to nitric oxide inhalation in early acute lung injury.

8. Puybasset L, Rouby JJ, Mourgeon E, et al: Factors influencing cardiopulmonary effects of

9. The neonatal inhaled nitric oxide study group : Inhaled nitric oxide in full-term and nearly

full-term infants with hypoxic respiratory failure. N Engl J Med 1997;336:597-604

10. Roberts JD, Fineman JR, Morin III FC, et al: Inhaled nitric oxide and persistent pulmonary

11. Davidson D, Barefield ES, Kattwinkel J, et al: Inhaled nitric oxide for the early treatment

-masked, placebo-controlled, dose-response, multicenter study. The I-NO/PPHN Study

13. Russel IA, Zwass MS, Fineman JR, et al: The effects of inhaled nitric oxide on

postoperative pulmonary hypertension in infants and children undergoing surgical repair of

14. Koelling TM, Kirmse M, Di Salvo TG, et al: Inhaled nitric oxide improves exercise

capacity in patients with severe heart failure and right ventricular dysfunction.

15. Katayama Y, Higenbottam TW, Diaz de Atauri MJ, et al: Inhaled nitric oxide and arterial

16. Steudel W, ScherrerCrosbie M, Bloch KD et al: Sustained pulmonary hypertension and

right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of

17. Imanaka H, Hess D, Kirmse M, et al: Inaccuracies of nitric oxide delivery systems during

18. Lindberg L, Rydgren G: Production of nitric oxide in a delivery system for inhalation

of nitric oxide: a new equation for calculation. Br J Anaesth 1998; 80: 213-217

19. Cuthbertson BH, Dellinger P, Dyar OJ, et al: UK guidelines for the use of inhaled nitric

20. Luhr O, Frostell CG, Lönnqvist P-A, Heywood R: Induction of chromosome aberrations in

21. Schedin U, Norman M, Gustafsson LE, Herin P, Frostell CG: Endogenous nitric oxide

in the upper airways of healthy newborn infants. Pediatr Res 1996; 40: 148-151

22. Putensen C, Hörmann C, Kleinsasser A, et al: Cardiopulmonary effects of aerosolized

prostaglandin E₁ and nitric oxide inhalation in patients with acute respiratory distress

vasodilation induced by aerosolized zaprinast. Anesthesiology 1998; 88: 410-416.

24. Keh D, Gerlach M, Hurer I, Falke KJ, Gerlach H: Reduction of platelet trapping in

The importance of nitric oxide in the physiology of erection has been well established and recently reviewed (1). Nitric oxide is a potent, diatomic molecule that causes the endothelium-dependent and neurogenically mediated relaxation of vascular and trabecular smooth muscle in the penis. Studies by Ignarro et al, 1990 ; Kimoto et al, 1990; Bush et al, 1992, Azadzoi et al, 1990; Kim et al 1993; Burnett et al, 1992 (2-7) pioneered the elucidation of this relatively transient, but biologically powerful, radical as the key agent producing the complex physiological response of erection.

Schematic illustration of events required to produce an erection is shown in figures 1a. and 1b . This hypothetical mechanism involves activation of the efferent autonomic nerves, which results in dilation of the cavernosal and helicine arteries. The rapid increase in inflow of blood into the lacunar spaces is concurrent with a relaxation of the trabecular smooth muscle. This relaxation of the trabecular smooth muscle allows the lacunar spaces to fully dilate and fill with blood. As they fill and expand the increase in pressure causes the trabecular muscle to be forced against the tunica albuginia. The tunica albuginia is a rigid tissue that constrains the degree of expansion of the lacunar space. As a consequence of compression against the tunica albuginia, subtunical venules become `flattened' and a physically obstructive reduction in outflow of blood occurs. This simultaneous increase in outflow resistance, along with an increased inflow and relaxed lacunar smooth muscles produces an erection.

The pharmacological treatment of erectile dysfunction is somewhat complex because there is a diverse concert of physiological events needed to be accomplished for a successful erection to occur. Along with this multiplicity of events in the erection process, it stands to reason there is an equal multiplicity of sites of possible failure. Failure of just one alone or several may lead to mild to severe forms of erectile dysfunction. In general it appears that multiple pharmacological treatments are required therapy. Erectile dysfunction is clinically evaluated by the intracorporal injection of a mixture of drugs. This mixture includes an alpha receptor antagonist (to promote relaxation of the arterial inflow), a smooth muscle relaxant (to relax the lacunar and trabecular smooth muscles) and a phosphodiesterase inhibitor (to potentiate the direct smooth muscle relaxant action). This tri-mix mostly comprises a solution mixture of different amounts of phentolamine, PGE₁ and papavarine.

More recently the treatment of erectile dysfunction has undergone profound changes. The introduction of a new drug delivery system (MUSE) and approval of a new oral drug (Viagra) has accented impotence and created significant public awareness. Recent figures indicate erectile dysfunction is far more prevalent than earlier documented. In the United States it is estimated there are over 1 million new cases of mild to moderate impotence occurring each year. The MMAS survey (8) estimated a total of 7-20 million men suffer with erectile dysfunction in the US. World-wide the number is estimated at 170 million, mostly over the age of 50 years.

Until less than a year ago, approved treatment of erectile dysfunction involved either the intracorporal injection of PGE₁ (Caverject; Viridal/EDEX), or the transurethral delivery of PGE₁ (MUSE). In both cases drug efficacy and product convenience are not optimal. Response rates reported vary but they range between 50-80% for caverject and 25-70% for MUSE. In addition PGE₁ activates the nociception of pain which adds to the product inconvenience profile. The recent introduction of Viagra however has revolutionized treatment. It is an oral drug and based on sales figures is the most successful new drug introduced into the US market. Response rates to Viagra are slightly higher than locally administrated PGE₁-based therapy and it is a huge increase in product convenience. None the less it remains less than optimal, with overall response rates estimated between 50-70%. This may reflect the fact that a single pharmacological mechanism of action cannot adequately affect all the potential sites of dysfunction which can cause erectile failure.

The efficacy of drugs like Viagra supports the importance of nitric oxide in the erectile response. Viagra is a potent and selective inhibitor of type 5 phosphodiesterase which is found mainly in corporal smooth muscles. The role of PDE is to promote the degradation of cGMP since alterations in intracellular levels of cGMP regulate cytosolic Ca⁺⁺ levels and the contractile state of the muscle. The inhibition of the PDE prevents the degradation of cGMP, thereby promoting the reduction of intracellular calcium and causing smooth muscle relaxation. Drugs like Viagra act to potentiate the actions of nitric oxide since nitric oxide is the agent that stimulates guanylate cyclase leading to elevated levels of cGMP. It should be noted that in normal situations the release of nitric oxide is controlled by the autonomic nervous system. Viagra does not stimulate the release of nitric oxide. The action of PDE inhibitors is illustrated in Figure 2.

Understanding the importance of nitric oxide in the erectile process, along with recognizing that multi-pharmacological treatments may be needed to fully treat erectile dysfunction has led to the creation of some novel classes of drugs. In our laboratories we have focused on the synthesis of new molecules by chemically altering existing drugs so that they become nitric oxide donors while retaining the parent drug action. Compounds with dual pharmacological action that have been synthesized belong to the class known as alpha receptor antagonists. An example of this new class of molecules is NMI-221 or nitrosylated moxisylyte. Its structure is shown in Figure 3.

The rationale behind the nitrosylation of alpha antagonists is the concept that both increased blood inflow via dilation of cavernosal and helicine arteries and relaxation of trabecular and lacunar smooth muscles are required for a full erection. Alpha receptor antagonists promote vascular dilatation while nitric oxide promotes relaxation of both vascular and cavernosal smooth muscles. The effects of nitrosylated moxisylyte compared with moxisylyte alone on isolated corporal smooth muscle strips are illustrated in Figure 4. As can be seen there is a significant increase in the relaxant potency of moxisylyte following nitrosylation. Biochemical evidence for successful donation of nitric oxide into cavernosal smooth muscle cells is shown in Figure 5. Elevated tissue cGMP levels are measured following incubation with the nitrosylated moxisylyte derivative compared with either control or moxisylyte incubated tissues.

Finally, the effects of nitrosylation are illustrated in anesthetized rabbits in Figures 6 & 7. The enhanced efficacy (increase in intracorporal pressure) and duration of action following nitrosylation is clearly illustrated by the intracorporal injection of these drugs. There is no associated reduction of systemic blood pressure indicating either little `leakage' of active drug into the systemic circulation from the cavernosum or the total dose of drug administered is insufficient to effect blood pressure.

Enhancement of the nitric oxide pathway in the erectile response can also be achieved by the nitrosylation of molecules that inhibit phosphodiesterases. This may especially be demonstrable in men with erectile dysfunction, in whom a deficiency of endogenous nitric oxide synthesis occurs. In addition, local delivery of such drugs may reduce the potential of drug-induced side effects and increase the pharmacological response time.

In summary, endogenous nitric oxide production is required for successful penile erection. New therapies for the treatment of erectile dysfunction have therefor centered on modulation of the endogenous nitric oxide - guanylate cyclase pathway. Following the successful introduction of drugs that selectively inhibit phosphodiesterases and thereby elevate endogenous cGMP levels, the next generation of drugs may extend this concept into dual-acting pharmacology. Retaining the actions of nitric oxide while adding an additional mechanism of action (such as alpha receptor blockade or inhibition of phosphodiesterases) into a single molecule may provide an even more effective therapy. In addition it should be noted that these drugs might also be applicable to treat female sexual arousal dysfunction.

1. Burnett, A.L. Role of Nitric Oxide in the Physiology of erection. Biology of Reproduction 52, 485-489 (1995)

2. Ignarro, L. J., Bush. P.A., Buga, G.M., Wood, K.S., Fukuto, J.M. and Raifter, J. Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cabernosum smooth muscle. Biochem. Biophys. Res. Commun. 170, 843-850, (1990)

3. Kimoto,Y., Kessler,R., Constantinou,C.E. Endothelium dependent relaxation of human corpus cavernosum by bradykinin. J. Urology 144, 1015-1017, (1990)

4. Bush, P.A., Aronson,W.J., Buga, G.M., Raifer, J. and Ignarro, L.J. Nitric oxide is a potent relaxant of human and rabbit corpus cavernosum. J. Urology 147, 1650-1655 (1992)

5. Azadzoi, K.M., Kim, N., Brown, M.L, Goldstein, I., Cohen, R.A. and Saenz de Tejada, I. Endothelium-derived nitric oxide and cyclooxygenase products modulate corpus cavernosumsmooth muscle tone. J. Urology 147, 220-225 (1992)

6. Kim, N., Vardi, Y., Padma-Nathan, H., Daley, J., Goldstein, I. and Saenz de Tejada, I. Oxygen tension regulates the nitric oxide pathway: physiological role in penile erection J. Clin. Invest. 91, 437-443 (1993)

7. Burnett, A.L., Lowenstein, C.J., Bredt, D.S., Chang, T.S.K. and Snyder, S.H. Nitric Oxide: a physiologic mediator of penile erection. Science 257, 401-403 (1992)

8. Johannes, C.B., Araujo, A.B., Feldman, H.A., Derby, C.A. and McKinlay, J.B. Incidence of erectile dysfunction in aging men: Longitudinal results from the Massachusetts Male Aging Study (MMAS). Intl. J. Impotence Res. 10, Suppl. 3, Abst 414 (1998)

Nitric oxide (NO) releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs), are a class of recently described of NSAID derivatives generated by adding an nitroxybutyl moiety through an ether linkage to the parental NSAID. (1-4) These compounds exhibit a markedly reduced gastrointestinal toxicity, while retaining the anti-inflammatory and antipyretic activity of parent NSAID. Although NO-derived NSAIDs spare the gastric mucosa, they inhibit prostaglandin generation and exert a powerful anti-inflammatory effect. Indeed, preliminary animal studies indicate that NO-derived NSAIDs are more effective than conventional NSAIDs in reducing inflammation and pain in arthritic rats.

Caspases, a growing family of cysteine proteases related to the interleukin 1( (IL-1() converting enzyme (ICE) play a major role in transducing death signals in apoptosis. Caspases share sequence homology with Ced-3, a gene essential for apoptosis of the nematode Caenorhabditis elegans. (5) Activation of ICE-like cysteine proteases is now recognized to be a typical hallmark of apoptosis not only in nematodes, but also in mammalian cells There are at presently 10 to 15 human homologues of the Ced-3+cysteine protease. (6,7) Although all caspases cleave their substrate after an aspartic residue, their molecular structures suggest that the caspase family falls into two major groups: those that most resemble ICE (caspase-1) and those that most resemble CPP 32/Yama (caspase-3).(5-7) Caspase-1 denotes the original ICE and has the greatest specificity for cleaving pro-IL-1( (Figure 1, panel A).(7,8,9) Relevant for inflammation is the fact that other caspases and particularly those cleaving intracellular proteins involved in apoptosis either did not cleave the proIL-1( or required a 100-fold greater concentration compared with ICE.(8)

There is evidence that NO-donors, such as sodium nitroprusside, (9) inhibit caspases activation in vitro by causing the S-nitrosylation of thiol groups located in the enzyme catalytic core. (9) Previous studies carried out with purified subunit of caspase 1 and 3 demonstrates that the p17 subunit of CPP32 and the p20 subunit of caspase 1 are selective target for NO compounds, and that the S-nitrosylation of these subunits leads to a concentration-dependent inhibition of enzyme activity. (9) In a recent study by the 163+cysteine residue located in the active center of caspase 3.

Since ICE inhibition would be therapeutic in inflammation the effect of NO-derived NSAIDs on ICE activity and cytokine release was investigated in a mouse model of acute of acute inflammation. ICE bodes well as an ideal target for anti-inflammatory agents, since it plays a role in inflammation and only a minor role in apoptosis.

We have tested the effect of an NO-aspirin derivative, (2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester [NCX4016]) in a mouse animal model of acute liver failure induced by acute and generalized release of T cell-dependent cytokine ("T-cell mediated cytokine-related syndrome") (Figure 1, panel B). In this experimental model, liver damage is induced by in vivo administration of the lectin plant concanavalin A (con A). The so called Con A-induced hepatitis is characterized by focal liver cell apoptosis, massive liver infiltration by CD4+ cells and release of cytokines involved in cell to cell communication and inflammation, particularly, TNF(, IL-1(, IFN(, IL-2 and IL-6. Killing of liver cells by activated T lymphocytes or cytokine is mediated by crosslinking of the Fas and/or TNF-R1 on liver cells. Administration of NO-aspirin, but not aspirin, dose-dependently prevented IL-1(, and IFN( secretion (Figure 2, panel A and B) and protected from acute liver injury as measured by plasma aminotransferase levels (Figure 2, panel C) and liver histology (Figure 2, panel D-F), as well as DNA fragmentation and caspase 3 assay (data not shown). Moreover, NCX-4016 prevented Fas/FasL upregulation induced by Con A (Figure 2, panel G). By using western blot analysis on spleen lymphocytes we also demonstrated that NCX-4016 directly inhibited con A-induced ICE-activation (data not shown). Administration of sodium nitroprusside (SNP) simultanoeously with Con A significantly increased the rate of mice deaths (data not shown).

Taken together these data indicate that in vivo NO-NSAIDs administration inhibits caspase 1 activity and prevent cytokine release induced by inflammation. These results may have important therapeutic implications for treatment of inflammatory disorders since activation of caspase 1 is a limiting step in the process of maturation of cytokines critically involved in the initiation and amplification of inflammatory response. In this context, inhibition of the caspase 1 branch of cysteine protease family represents an ideal target for anti-inflammatory compounds.

1. Wallace JL, Reuter B, Cicala C, McKnight W, Grisham MB, Cirino G. Novel nonsteroidal anti-inflammatory drug derivatives with markedly reduced ulcerogenic properties in the rat. Gastroenterology 1994; 107: 173-197.

2. Fiorucci S, Antonelli E, Migliorati G, Santucci L, Morelli O, Federici B, A.Morelli. TNF(-processing enzyme inhibitors prevent aspirin-induced TNF( release and protect against mucosal injury in rats. Alim Pharmacol Ther, in press.

3. Fiorucci, S, Ea Antonelli, L Santucci, O Morelli, M Miglietti, B Federici, P Del Soldato, A Morelli NO-NSAID Gastrointestinal Safety is Related to Inhibition of ICE-like Cysteine Proteases. New Insights Into the Molecular Basis of NSAID Gastropathy. Gastroenterology, 1998 (Abs)

4. Fiorucci S, L Santucci, B Federici, E Antonelli, E Distrutti, O Morelli, P del Soldato, A. Morelli Nitric Oxide (NO)-releasing NSAIDs Inhibit Interleukin-1( Converting Enzyme(ICE)-Like Cysteine Proteases and Protect Endothelial Cells From Apoptotosis Induced by TNF(. Alim Pharmacol Ther, in press

5) Ashkenazi A, VM Dixit. Death receptors: signaling and modulation. Science 281: 1305-1308.

6) Salvesen GS, VM. Dixit. Caspases: intracellular signaling by proteolysis. Cell 1997; 91:443-446.

7) Thornberry NA, Y Lazebbnik. Caspases: enemies within. Science 1998; 281: 1312-1316.

8) Okamura, H., H. Tsutsui, T. Komatsu, M. Yutsudo, A. Hakura, T. Tanimoto, K. Torigoe, T.Okura, Y. Nukada, K. Hattori, et al. Cloning of a new cytokine that induces IFN-gamma production by T-cells. Nature. 1995; 378:88-91.

Nitric oxide via inhibition of interleukin-1 converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J Exp Med 1997; 185: 601-607.

Figure 1. Panel A. Modality of caspase activation. Panel B. Structure of NCX-4016 (No-releasing aspirin).

Figure 2. Panel A and B Time-course of cytokines (IL-1( and IFN() in animals treated with Con A alone or Con A + NCX-4016 75 mg/kg. Con A was administered i.v. and NCX-4016 i.p. simultaneously with Con A. Data are mean ( SE of 6 mice. IL-1( and IFN( have been measured by using a commercial ELISA kit (DuoSet(. Genzyme, Boston, MA). Panel C. Time-course of aminotranserases (AST and ALT) plasma levels in animals treated with Con A alone or Con A + NCX-4016 75 mg/kg. Data are mean ( SE of 6 mice. Panel D-F. Histologic detection of liver damage in mice treated with Con A. Panel D. Liver section from animals treated with Con + NCX-4016 10 mg/kg. Note extensive liver injury and apoptosis (square). H&E staining. Magnification 200 x. Panel E. NCX-4016 protects from damage induced by Con A. Small necrotic areas are seen (square) Magnification 100 x. Panel G. NCX-4016 75 mg/kg markedly reduces liver injury induced by Con A. Magnification 100 x. Panel H. NCX-4016, prevents Con A-induced Fas/FasL upregulation on spleen lymphocytes. Data are mean ( SE of 6 mice.

It must have been gratifying for Boris Vargaftig to introduce a nitric oxide conference less than three days after the announcement of the Nobel prize for nitric oxide discovery. Dr. Moncada (The Wolfson Institute, University College London) who inspired the nitric-oxide component of the work of most of the speakers (and audience) in Paris, had been highly instrumental in putting together the programme of speakers for this Euroconference and gave a memorable opening review lecture. The disappointment for the participants in this Euroconference was that Dr. Salvador Moncada, had not shared the Nobel prize.
Dr. Moncada's recent work examined in greater detail the mechanisms by which NO inhibits mitochondrial respiration. It shows that although NO inhibits complex IV in a reversible manner+; long-term exposure to NO results in a persistent inhibition of complex I that is concomitant with a reduction of intracellular reduced glutathione (GSH)+;This does not involve peroxynitrite (ONOO-) but seems to be due to S-nitrosylation of critical thiols in the enzyme complex since it is reversed by replenishment of intracellular GSH. These results suggests a stepwise process induced by NO from physiology (inhibition complex IV) to pathophysiology (inhibition complex I) reminiscent to some features observed in neurodegenerative diseases like Parkinson’s or Alzheimer’s diseases.

NO SYNTHASE

NO is formed from L-Arginine by a family of enzymes, the NO Synthase (NOS) which exist in three different isoforms+: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). The nNOS may regulate learning and memory formation in brain or contribute to neurodegenerative disorders, while eNOS promotes vascular relaxation and inhibits platelet agregation; both enzymes are constitutive and calcium dependent. The iNOS is induced by endotoxins and cytokines; overexpression may lead to tissue injury among other harmful effects.
All NOS are homodimeric and have reductase and highly conserved oxygenase polypeptide domains within each monomer. The reductase domain binds FAD, NADPH and FMN and the oxygenase domain binds heme, tetrahydrobiopterin (BH4). calmodulin linking the two domains . The crystal structure of mouse iNOS oxygenase domain was presented by Denis Stuehr( Cleveland USA). His group has carried out site directed mutagenesis and amino acid deletions to provide structure-function information about arginine binding, tetrahydrobiopterin in the dimer and the flavin-to- heme 'trans' intersubunit transfer of electrons in the heterodimer . He showed that domain swapping occurs in iNOS to properly align reductase and oxygenase domain for NO formation.
As presented by Bernd Mayer ( Graz, Austria),.BH4 binds with ease to one dimer, but slowly to the second. The physiological consequence is that NO, O2- and ONOO- will be formed (O2- from the BH4-free subunit). Superoxide dismutase(SOD) at high concentrations improves NO formation by reducing the peroxynitrite reaction. In the absence of SOD and the presence of GSH a storage form of NO may result (GSNO). If NOS is saturated with arginine, without BH4, the product will be hydrogen peroxide, which also stimulates cGMP formation, Flavin co-factor binding site inhibitors are less common than active site inhibitors. Pharmacological tools (anti-pterins) displace BH4 from NOS which unexpectedly did not lower basal NOS activity. The view of Harald Schmidt (W[cedilla]rzburg, Germany) is that basal nNOS activity may be independent of BH4, which may bind to the dimerisation domain, thereby preventing monomerization and increasing stability. There are pterin-based activators and inhibitors of NOS, useful tools to understand BH4 binding and to modulate enzyme activity.
William Sessa (Yale, USA) showed that eNOS is compartmentalized by myristoylation and palmitoylation to the Golgi area and to cholesterol- and glycolipid-rich regions of the plasma membrane called caveolae . This subcellular localization is critical for optimal NO production. The process of getting eNOS docked is subject to regulation by interaction with caveolin and by heat shock protein 90, whose inhibition lowers agonist-stimulated NO production. The activation of eNOS in response to receptor-agonist calcium elevation is transient compared to the activation by shear stress. Tyrosine kinase inhibitors lower stress-activated NOS, which is insensitive to calcium removal. Shear stress studies on eNOS showed tyrosine and serine residue phosphorylation occurred but did not account entirely for the calcium independency. According to Rudi Busse, ( Frankfurt , Germany) eNOS is part of a 'multimolecular complex' consisting of proteins which co-precipitate with eNOS, and are sensitive to tyrosine phosphorylation.

NO AND CELL DEATH

NO from NO-donors or from cytokine-induced iNOS is sufficient to cause DNA strand breaks. If ONOO- is formed, oxidized guanine and abasic sites contribute to single strand breaks, whereas if the nitrosating agent N2O3 is formed ,DNA cross links and deaminated bases give the same result. Steven Tannenbaum ( M.I.T Cambridge USA) hypothesized that DNA damage will arise from NO, depending on the dose, cell type, and cellular environment and will cause cells to undergo apoptosis or enhanced mutation. His lecture concentrated on NO chemistry, and how formation and action of damaging species ONOO-, N203, Fenton reaction ferryl species and hydroxyl radical may differ in cells placed at different distances from the site of NO production.
High concentrations of NO is potentially cytotoxic. Cell death induced by NO may be necrotic or apoptotic but NO may also signals cell protection. Apoptosis caused by cytokine and lipo-polysaccharide induced iNOS and protection by low dose NO was exquisitively demonstrated and discussed by Bernhard Br[cedilla]ne( Erlangen, Germany). He described treatment of RAW 264.7 macrophages with GSNO or cytokines leading to p53 tumour suppressor gene expression, caspase activation, , poly ADP ribose polymerase(PARP) cleavage and Bcl2 expression. Protection by low dose NO resulted when NFk[apple]B was increased, protective proteins such as HSP-70, haemoxygenase-1 and COX-2 as well NO/O2- .interactions and thiol modification of caspases, could favour protection from cell death.

TARGETS OF NO

A most important receptor for NO is soluble guanylate cyclase (sGC) which mediates many physiological actions of NO such as vasorelaxation, inhibition of platelet aggregation or synaptic transmission. The stimulatory effect of NO is mediated by interaction with the prosthetic heme group of sGC+. Doris Koesling (Berlin, Germany) presented a new mechanism of stimulation of sGC by using YC-1 (a benzylindazole derivative). This compound activates sGC independently of NO and of carbon monoxide. By binding to an allosteric site on sGC, YC-1 can sensitize sGC to its natural agonist and also to carbon monoxide.Also by exerting inhibitory effects on cGMP phosphodiesterases this compound can increase cGMP several hundred fold! Others favorite targets for NO are metalloenzymes that contain Fe-S clusters and/or redox sensitive sites. One of the best example was given by Jean-Claude Drapier (Gif-sur-Yvette, France) with the case of aconitase. The excellent lecture of Rafael Radi (Montevideo ,Uruguay) was on the biological reactions of ONOO-. the reaction product of NO and superoxide O2-. He showed how this highly reactive and cytotoxic molecule accounts for of O2- and NO-dependent toxicity. It should be noted that signalling for NO is diverse. Investigations of neuronal cell development and survival suggested activation of the GTP binding protein p21 Ras and its GTPase activity by NO-redox sensitive modification, resulting in activation of extracellular regulated kinase (ERK) (Ted M. Dawson,).

NO MODULATORS+: THERAPEUTIC AGENTS

The actions and reactions of NO suggest that it may exacerbate certain disease states+:stroke, Alzheimer's or Parkinson’s disease, Huntingdon’s chorea, septic shock, inflammatory airway disease including asthma - or be therapeutic, as for pulmonary hypertension,vascular disorders or male impotence. NO synthesised from eNOS seems to have exclusively physiological actions whereas NO from iNOS and nNOS may be pathologic. Selectivity among NOS isoforms is thus highly desirable for therapeutical purposes. NO acute toxicity in brain appears to require O2- with which it reacts to form ONOO-. Neurotoxicity could result from the inhibition of respiratory chain enzymes, and from its DNA damaging ability. Activation of the DNA repair enzyme PARP by NO/ONOO- results in loss of cellular NAD leading to a fatal depletion in energy stores. Ted . Dawson (Baltimore, USA) showed that in vitro inhibition of PARP attenuates toxicity in cortical cultures, while in vivo PARP knockout mice are resistant to focal ischemia and to the neurotoxin MPTP which reproduces features of Parkinson's disease+ Evaluation of the neuroprotective properties of NOS inhibitors and antioxidants were examined by Beaufour-Ipsen researchers. Interestingly, they found that combination of the two types of drugs exert a synergistic neuroprotective effect. For this reason ,they developped novel compounds possessing dual activity. Pierre-Etienne Chabrier ( les Ulis, France) described BN 80933, a selective nNOS inhibitor and a potent antioxidant+;This compound shows a remarkable efficacy in delayed post treatment in models of transient ischemia to reduce infarct volume and to improve behavioral recovery. Dr. Richard Knowles from Glaxo-Wellcome (Stevenage, UK) examined the pathologies associated with iNOS (septic shock and tumour growth,) which he has studied in depth,as well as the rôle of NO in autoimmune diseases and resistance to infection .He reported expression of iNOS in human gastric, CNS, breast and other tumours . Tumour growth in vivo in mouse was inhibited by 1400W, an iNOS selective inhibitor. Resolution of conditions like septic shock may entail precisely timed use of selective iNOS inhibitors since non selective NOS inhibitor such as L-NMMA exacerbate early microvascular leakage by affecting vascular eNOS. Peter J Barnes, (London,UK) measured exhaled NO and found vastly increased levels in patients with asthma, but not with all lung diseases. NO is not diagnostic for asthma since rises occur in inflammatory disease, and if nasal air contaminates the airways' sample. Inhaled NOS inhibitors and steroids reduce exhaled NO. Because of the multifactorial nature of inflammatory disease, NO can tip the balance of cytotoxic- and cytoprotective-cytokine producing lymphocytes. More advanced clinical studies were presented by Claes Frostel (Stockholm, Sweridge) showing the use of 0.1-100 parts per billion inhaled NO in hypoxic newborns or acute lung injury in adults in order to improve oxygenation. In a more prospective way, Victor Dzau ( Harvard, USA) introduced gene therapy strategies using either eNOS or iNOS and their potential benefit to treat vascular proliferative disorders. Dr Gordon Letts ( NITROMED, Bedford , USA) explained how Viagra, a phosphodiesterase V inhibitor, modulates the NO-cGMP system, but is ineffective in males not producing enough basal NO. Developing compounds containing "parent drug-inert linker-NO producer" components is relevant for the male impotence market, and may have a promising future in other therapies.

CONCLUSION

This Euroconference showed how dynamic is NO basic research and emphasized clearly its therapeutic potential . To complete the experience for registered participants, invited speakers had contributed to a conference proceedings book, reminiscent of a telephone directory, but much more interesting. Corresponding address:

Dr Irene C Green, Principal Research Fellow University of Sussex, Brighton BN1 9QG East Sussex, BN1 9QG UK tel +44 1273 678404 fax +44 1273 678433 email i.c.green@sussex.ac.uk

Monoxyde d'azote en physiopathologie

Nitric oxide: basic research and clinical applications

Résumés étendus

Extensive abstracts

Effects of NO on DNA Damage and DNA Synthesis

Dr. Dennis Stuehr

Regulation of endothelial-derived nitric oxide synthase by post-translational modifications and protein-protein interactions.

William C. Sessa, Associate Professor of Pharmacology, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06536

Structure and function of nitric oxide synthases

Bernd Mayer

Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria

Ca2+-independent activation of the endothelial nitric oxide synthase

Rudi Busse

Institut fur Kardiovaskulare Physiologie, Klinikum der J.W.G.-Universitat, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany

Pterins and anti-pterins in NO synthesis

Harald H.H.W. Schmidt, Lothar Fröhlich, Wolfgang Pfleiderer* and Peter Kotsonis

Department of Pharmacology and Toxicology, Julius-Maximilians-University, Versbacher Str. 9, D-97078 Würzburg, Germany; *Faculty of Chemistry, University of Konstanz, Germany

The Role of Nitric Oxide in Apoptosis

Bernhard Brüne, Andreas von Knethen, and Katrin Sandau

University of Erlangen-Nürnberg, Faculty of Medicine, Department of Medicine IV-

Experimental Division, Erlangen, Germany

Effects of NO on DNA Damage and DNA Synthesis

Steven R. Tannenbaum

Massachusetts Institute of Technology

Division of Bioengineering and Environmental Health

77 Massachusetts Avenue, Rm. 56-731A

Cambridge, MA 02139-4307, U.S.A.

BIOLOGICAL REACTIONS OF PEROXYNITRITE

Rafael Radi

Departamento de Bioquímica, Facultad de Medicina, Universidad de la

República, Montevideo, Uruguay

Switching enzymes off: the case of aconitases

Jean-Claude Drapier and Cécile Bouton

ICSN-CNRS, Avenue de la Terrasse 91190 Gif-sur-Yvette, France

Progress In Brain Research "1998"

Nitric Oxide, PARP and other Perpetrators Relevant to Stroke and Neurodegeneration.

Ted M. Dawson, M.D., Ph.D.

Department of Neurology and Neuroscience, Johns Hopkins University School of Medicine, , 600 North Wolfe Street - Carnegie 214, Baltimore, MD 21287

NITRIC OXIDE AND AIRWAY DISEASE

NITRIC OXIDE IN THE LUNGS

Peter Barnes

National Heart & Lung Institute, Imperial College, London, UK.

Inhaled nitric oxide: experimental and clinical experiences

Claes G Frostell, MD, PhD

PICU

Astrid Lindgren Children's Hospital

at Karolinska Hospital

S-171 76 Stockholm

Sweden

The role of Nitric Oxide in Impotence

NO-derived NSAIDs are a new class of anti-inflammatory agents that spare the gastric mucosa and inhibit cytokine release.

Fiorucci Stefano, M.D.*, and Piero del Soldato, Ph.D.**

Gastroenterology and hepathology section,

Department of Internal medicine

University of Perugia, Italy

** NicOx, Paris, France.